Antisense modulation of resistin expression

ABSTRACT

Antisense compounds, compositions and methods are provided for modulating the expression of resistin. The compositions comprise antisense compounds, particularly antisense oligonucleotides, targeted to nucleic acids encoding resistin. Methods of using these compounds for modulation of resistin expression and for treatment of diseases associated with expression of resistin are provided.

FIELD OF THE INVENTION

[0001] The present invention provides compositions and methods formodulating the expression of resistin. In particular, this inventionrelates to compounds, particularly oligonucleotides, specificallyhybridizable with nucleic acids encoding resistin. Such compounds havebeen shown to modulate the expression of resistin.

BACKGROUND OF THE INVENTION

[0002] Insulin resistance is defined as a failure of target tissues(adipose, liver, skeletal and cardiac muscle) to respond normally toinsulin. At the molecular level, this resistance can occur anywhere inthe insulin signaling pathway, from receptor-binding to downstreamsignaling events. Obesity-associated insulin resistance is manifested byincreased hepatic glucose output and reduced glucose disposal inperipheral tissues. Quantitatively, skeletal muscle is the mostimportant site of insulin-mediated glucose disposal. Adipose tissueclearly plays a significant role in the pathogenesis of insulinresistance, as shown by the high correlation between obesity and insulinresistance. Obesity-induced insulin resistance is affected both by thetotal amount of adipose tissue and its distribution. Both visceral anddeep subcutaneous adipose tissues are associated with insulinresistance.

[0003] Excessive free fatty acids (FFAS) released by lipolysis fromadipose tissue have been implicated in non-insulin dependent diabetesmellitus. FFAs have a deleterious effect on insulin uptake by the liverand contribute to the increased hepatic glucose release. The observationthat adipose-specific changes in gene expression alter insulinsensitivity in muscle and liver in the mouse reinforces the concept thatsignals emanating from adipose tissue regulate glucose homeostasis(Steppan and Lazar, Trends Endocrinol Metab, 2002, 13, 18-23).

[0004] Resistin, a gene named for its role in insulin resistance, isalso known as FIZZ3 (found in inflammatory zone 3). It was identifiedand localized to chromosome 19p13.3 (Kim et al., J. Biol. Chem., 2001,276, 11252-11256; Steppan et al., Nature, 2001, 409, 307-312).

[0005] An isolated nucleic acid sequence encoding a mammalian resistinis disclosed and claimed in PCT publication WO 00/64920 (Lazar, 2000).Resistin mRNA, which encodes a 114-amino acid polypeptide containing a20-amino acid signal sequence, is induced during 3T3-L1 adipogenesis ofwhite adipose tissue in both the mouse and rat The highest levels ofexpression are observed in female gonadal adipose tissue (Steppan andLazar, Trends Endocrinol Metab, 2002, 13, 18-23).

[0006] Investigations of resistin expression in human adipocytes haveindicated that the increased resistin expression observed in abdominalfat, relative to other types of adipose tissue, may explain theincreased risk of type 2 diabetes associated with central obesity(McTernan et al., Lancet, 2002, 359, 46-47).

[0007] Regulators of resistin have been employed in investigations ofits biological roles. Steppan et al. have found that circulatingresistin levels are decreased by the anti-diabetic drug rosiglitazone (amember of the thiazolidinedione class of class of anti-diabetic drugs)and increased in diet-induced and genetic forms of obesity in mice. Inaddition, treatment of normal mice with recombinant resistin impairsglucose tolerance and insulin action. The conclusion of this study isthat resistin is a hormone that potentially links obesity to diabetes(Steppan et al., Nature, 2001, 409, 307-312).

[0008] Expression levels of resistin in mice and in 3T3-L1 adipocyteshave been found to be induced by beta-3 adrenergic agonists anddown-regulated by tumor necrosis factor-alpha and isoproterenol(Fasshauer et al., FEBS Lett., 2001, 500, 60-63; Fasshauer et al.,Biochem. Biophys. Res. Commun., 2001, 288, 1027-1031; Martinez et al.,J. Physiol. Biochem., 2001, 57, 287-288).

[0009] The potential involvement of resistin in insulin resistanceindicates that it may prove to be a useful target for therapeuticintervention in type 2 diabetes and/or other disorders related toobesity.

[0010] Disclosed and claimed in PCT publication WO 00/64920 is ananti-diabetic composition comprising a pharmaceutically acceptablecarrier and an isolated nucleic acid sequence complementary to saidmammalian resistin sequence, in the antisense direction, and a method ofalleviating type 2 diabetes via administration of said anti-diabeticcomposition to a patient afflicted with type 2 diabetes (Lazar, 2000).

[0011] Currently, there are no known therapeutic agents that effectivelyinhibit the synthesis of resistin. To date, investigative strategiesaimed at modulating resistin expression have involved the use ofrosiglitazone, beta-3 adrenergic agonists, tumor necrosis factor-alphaand isoproterenol. Consequently, there remains a long felt need foradditional agents capable of effectively inhibiting resistin function.

[0012] Antisense technology is emerging as an effective means forreducing the expression of specific gene products and may thereforeprove to be uniquely useful in a number of therapeutic, diagnostic, andresearch applications for the modulation of expression of resistin.

[0013] The present invention provides compositions and methods formodulating expression of resistin.

SUMMARY OF THE INVENTION

[0014] The present invention is directed to compounds, particularlyantisense oligonucleotides, which are targeted to a nucleic acidencoding resistin, and which modulate the expression of resistin.Pharmaceutical and other compositions comprising the compounds of theinvention are also provided. Further provided are methods of modulatingthe expression of resistin in cells or tissues comprising contactingsaid cells or tissues with one or more of the antisense compounds orcompositions of the invention. Further provided are methods of treatingan animal, particularly a human, suspected of having or being prone to adisease or condition associated with expression of resistin byadministering a therapeutically or prophylactically effective amount ofone or more of the antisense compounds or compositions of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0015] The present invention employs oligomeric compounds, particularlyantisense oligonucleotides, for use in modulating the function ofnucleic acid molecules encoding resistin, ultimately modulating theamount of resistin produced. This is accomplished by providing antisensecompounds which specifically hybridize with one or more nucleic acidsencoding resistin. As used herein, the terms “target nucleic acid” and“nucleic acid encoding resistin” encompass DNA encoding resistin, RNA(including pre-mRNA and mRNA) transcribed from such DNA, and also cDNAderived from such RNA. The specific hybridization of an oligomericcompound with its target nucleic acid interferes with the normalfunction of the nucleic acid. This modulation of function of a targetnucleic acid by compounds which specifically hybridize to it isgenerally referred to as “antisense”. The functions of DNA to beinterfered with include replication and transcription. The functions ofRNA to be interfered with include all vital functions such as, forexample, translocation of the RNA to the site of protein translation,translocation of the RNA to sites within the cell which are distant fromthe site of RNA synthesis, translation of protein from the RNA, splicingof the RNA to yield one or more mRNA species, and catalytic activitywhich may be engaged in or facilitated by the RNA. The overall effect ofsuch interference with target nucleic acid function is modulation of theexpression of resistin. In the context of the present invention,“modulation” means either an increase (stimulation) or a decrease(inhibition) in the expression of a gene. In the context of the presentinvention, inhibition is the preferred form of modulation of geneexpression and mRNA is a preferred target.

[0016] It is preferred to target specific nucleic acids for antisense.“Targeting” an antisense compound to a particular nucleic acid, in thecontext of this invention, is a multistep process. The process usuallybegins with the identification of a nucleic acid sequence whose functionis to be modulated. This may be, for example, a cellular gene (or mRNAtranscribed from the gene) whose expression is associated with aparticular disorder or disease state, or a nucleic acid molecule from aninfectious agent. In the present invention, the target is a nucleic acidmolecule encoding resistin. The targeting process also includesdetermination of a site or sites within this gene for the antisenseinteraction to occur such that the desired effect, e.g., detection ormodulation of expression of the protein, will result. Within the contextof the present invention, a preferred intragenic site is the regionencompassing the translation initiation or termination codon of the openreading frame (ORF) of the gene. Since, as is known in the art, thetranslation initiation codon is typically 5′-AUG (in transcribed mRNAmolecules; 5′-ATG in the corresponding DNA molecule), the translationinitiation codon is also referred to as the “AUG codon,” the “startcodon” or the “AUG start codon”. A minority of genes have a translationinitiation codon having the RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and5′-AUA, 5′-ACG and 5′-CUG have been shown to function in vivo. Thus, theterms “translation initiation codon” and “start codon” can encompassmany codon sequences, even though the initiator amino acid in eachinstance is typically methionine (in eukaryotes) or formylmethionine (inprokaryotes). It is also known in the art that eukaryotic andprokaryotic genes may have two or more alternative start codons, any oneof which may be preferentially utilized for translation initiation in aparticular cell type or tissue, or under a particular set of conditions.In the context of the invention, “start codon” and “translationinitiation codon” refer to the codon or codons that are used in vivo toinitiate translation of an mRNA molecule transcribed from a geneencoding resistin, regardless of the sequence(s) of such codons.

[0017] It is also known in the art that a translation termination codon(or “stop codon”) of a gene may have one of three sequences, i.e.,5′-UAA, 5′-UAG and 5′-UGA (the corresponding DNA sequences are 5′-TAA,5′-TAG and 5′-TGA, respectively). The terms “start codon region” and“translation initiation codon region” refer to a portion of such an mRNAor gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5′ or 3′) from a translationinitiation codon. Similarly, the terms “stop codon region” and“translation termination codon region” refer to a portion of such anmRNA or gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5′ or 3′) from a translationtermination codon.

[0018] The open reading frame (ORF) or “coding region,” which is knownin the art to refer to the region between the translation initiationcodon and the translation termination codon, is also a region which maybe targeted effectively. Other target regions include the 5′untranslated region (5′UTR), known in the art to refer to the portion ofan mRNA in the 5′ direction from the translation initiation codon, andthus including nucleotides between the 5′ cap site and the translationinitiation codon of an mRNA or corresponding nucleotides on the gene,and the 3′ untranslated region (3′UTR), known in the art to refer to theportion of an mRNA in the 3′ direction from the translation terminationcodon, and thus including nucleotides between the translationtermination codon and 3′ end of an mRNA or corresponding nucleotides onthe gene. The 5′ cap of an mRNA comprises an N7-methylated guanosineresidue joined to the 5′-most residue of the mRNA via a 5′-5′triphosphate linkage. The 5′ cap region of an mRNA is considered toinclude the 5′ cap structure itself as well as the first 50 nucleotidesadjacent to the cap. The 5′ cap region may also be a preferred targetregion.

[0019] Although some eukaryotic mRNA transcripts are directlytranslated, many contain one or more regions, known as “introns,” whichare excised from a transcript before it is translated. The remaining(and therefore translated) regions are known as “exons” and are splicedtogether to form a continuous mRNA sequence. mRNA splice sites, i.e.,intron-exon junctions, may also be preferred target regions, and areparticularly useful in situations where aberrant splicing is implicatedin disease, or where an overproduction of a particular mRNA spliceproduct is implicated in disease. Aberrant fusion junctions due torearrangements or deletions are also preferred targets. mRNA transcriptsproduced via the process of splicing of two (or more) mRNAs fromdifferent gene sources are known as “fusion transcripts”. It has alsobeen found that introns can be effective, and therefore preferred,target regions for antisense compounds targeted, for example, to DNA orpre-mRNA.

[0020] It is also known in the art that alternative RNA transcripts canbe produced from the same genomic region of DNA. These alternativetranscripts are generally known as “variants”. More specifically,“pre-mRNA variants” are transcripts produced from the same genomic DNAthat differ from other transcripts produced from the same genomic DNA ineither their start or stop position and contain both intronic andextronic regions.

[0021] Upon excision of one or more exon or intron regions or portionsthereof during splicing, pre-mRNA variants produce smaller “mRNAvariants”. Consequently, mRNA variants are processed pre-mRNA variantsand each unique pre-mRNA variant must always produce a unique mRNAvariant as a result of splicing. These mRNA variants are also known as“alternative splice variants”. If no splicing of the pre-mRNA variantoccurs then the pre-mRNA variant is identical to the mRNA variant.

[0022] It is also known in the art that variants can be produced throughthe use of alternative signals to start or stop transcription and thatpre-mRNAs and mRNAs can possess more that one start codon or stop codon.Variants that originate from a pre-mRNA or mRNA that use alternativestart codons are known as “alternative start variants” of that pre-mRNAor mRNA. Those transcripts that use an alternative stop codon are knownas “alternative stop variants” of that pre-mRNA or mRNA. One specifictype of alternative stop variant is the “polyA variant” in which themultiple transcripts produced result from the alternative selection ofone of the “polyA stop signals” by the transcription machinery, therebyproducing transcripts that terminate at unique polyA sites.

[0023] Once one or more target sites have been identified,oligonucleotides are chosen which are sufficiently complementary to thetarget, i.e., hybridize sufficiently well and with sufficientspecificity, to give the desired effect.

[0024] In the context of this invention, “hybridization” means hydrogenbonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteenhydrogen bonding, between complementary nucleoside or nucleotide bases.For example, adenine and thymine are complementary nucleobases whichpair through the formation of hydrogen bonds. “Complementary,” as usedherein, refers to the capacity for precise pairing between twonucleotides. For example, if a nucleotide at a certain position of anoligonucleotide is capable of hydrogen bonding with a nucleotide at thesame position of a DNA or RNA molecule, then the oligonucleotide and theDNA or RNA are considered to be complementary to each other at thatposition. The oligonucleotide and the DNA or RNA are complementary toeach other when a sufficient number of corresponding positions in eachmolecule are occupied by nucleotides which can hydrogen bond with eachother. Thus, “specifically hybridizable” and “complementary” are termswhich are used to indicate a sufficient degree of complementarity orprecise pairing such that stable and specific binding occurs between theoligonucleotide and the DNA or RNA target. It is understood in the artthat the sequence of an antisense compound need not be 100%complementary to that of its target nucleic acid to be specificallyhybridizable.

[0025] An antisense compound is specifically hybridizable when bindingof the compound to the target DNA or RNA molecule interferes with thenormal function of the target DNA or RNA to cause a loss of activity,and there is a sufficient degree of complementarity to avoidnon-specific binding of the antisense compound to non-target sequencesunder conditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays or therapeutictreatment, and in the case of in vitro assays, under conditions in whichthe assays are performed. It is preferred that the antisense compoundsof the present invention comprise at least 80% sequence complementarityto a target region within the target nucleic acid, moreover that theycomprise 90% sequence complementarity and even more comprise 95%sequence complementarity to the target region within the target nucleicacid sequence to which they are targeted. For example, an antisensecompound in which 18 of 20 nucleobases of the antisense compound arecomplementary, and would therefore specifically hybridize, to a targetregion would represent 90 percent complementarity. Percentcomplementarity of an antisense compound with a region of a targetnucleic acid can be determined routinely using basic local alignmentsearch tools (BLAST programs) (Altschul et al., J. Mol. Biol., 1990,215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656).

[0026] Antisense and other compounds of the invention, which hybridizeto the target and inhibit expression of the target, are identifiedthrough experimentation, and representative sequences of these compoundsare hereinbelow identified as preferred embodiments of the invention.The sites to which these preferred antisense compounds are specificallyhybridizable are hereinbelow referred to as “preferred target regions”and are therefore preferred sites for targeting. As used herein the term“preferred target region” is defined as at least an 8-nucleobase portionof a target region to which an active antisense compound is targeted.While not wishing to be bound by theory, it is presently believed thatthese target regions represent regions of the target nucleic acid whichare accessible for hybridization.

[0027] While the specific sequences of particular preferred targetregions are set forth below, one of skill in the art will recognize thatthese serve to illustrate and describe particular embodiments within thescope of the present invention. Additional preferred target regions maybe identified by one having ordinary skill.

[0028] Target regions 8-80 nucleobases in length comprising a stretch ofat least eight (8) consecutive nucleobases selected from within theillustrative preferred target regions are considered to be suitablepreferred target regions as well.

[0029] Exemplary good preferred target regions include DNA or RNAsequences that comprise at least the 8 consecutive nucleobases from the5′-terminus of one of the illustrative preferred target regions (theremaining nucleobases being a consecutive stretch of the same DNA or RNAbeginning immediately upstream of the 5′-terminus of the target regionand continuing until the DNA or RNA contains about 8 to about 80nucleobases). Similarly good preferred target regions are represented byDNA or RNA sequences that comprise at least the 8 consecutivenucleobases from the 3′-terminus of one of the illustrative preferredtarget regions (the remaining nucleobases being a consecutive stretch ofthe same DNA or RNA beginning immediately downstream of the 3′-terminusof the target region and continuing until the DNA or RNA contains about8 to about 80 nucleobases). One having skill in the art, once armed withthe empirically-derived preferred target regions illustrated herein willbe able, without undue experimentation, to identify further preferredtarget regions. In addition, one having ordinary skill in the art willalso be able to identify additional compounds, including oligonucleotideprobes and primers, that specifically hybridize to these preferredtarget regions using techniques available to the ordinary practitionerin the art.

[0030] Antisense compounds are commonly used as research reagents anddiagnostics. For example, antisense oligonucleotides, which are able toinhibit gene expression with exquisite specificity, are often used bythose of ordinary skill to elucidate the function of particular genes.Antisense compounds are also used, for example, to distinguish betweenfunctions of various members of a biological pathway. Antisensemodulation has, therefore, been harnessed for research use.

[0031] For use in kits and diagnostics, the antisense compounds of thepresent invention, either alone or in combination with other antisensecompounds or therapeutics, can be used as tools in differential and/orcombinatorial analyses to elucidate expression patterns of a portion orthe entire complement of genes expressed within cells and tissues.

[0032] Expression patterns within cells or tissues treated with one ormore antisense compounds are compared to control cells or tissues nottreated with antisense compounds and the patterns produced are analyzedfor differential levels of gene expression as they pertain, for example,to disease association, signaling pathway, cellular localization,expression level, size, structure or function of the genes examined.These analyses can be performed on stimulated or unstimulated cells andin the presence or absence of other compounds which affect expressionpatterns.

[0033] Examples of methods of gene expression analysis known in the artinclude DNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000,480, 17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serialanalysis of gene expression) (Madden, et al., Drug Discov. Today, 2000,5, 415-425), READS (restriction enzyme amplification of digested cDNAs)(Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (totalgene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci.U.S.A., 2000, 97, 1976-81), protein arrays and proteomics (Celis, etal., FEBS Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis,1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis, etal., FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000,80, 143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal.Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41,203-208), subtractive cloning, differential display (DD) (Jurecic andBelmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomichybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31,286-96), FISH (fluorescent in situ hybridization) techniques (Going andGusterson, Eur. J. Cancer, 1999, 35, 1895-904) and mass spectrometrymethods (reviewed in To, Comb. Chem. High Throughput Screen, 2000, 3,235-41).

[0034] The specificity and sensitivity of antisense is also harnessed bythose of skill in the art for therapeutic uses. Antisenseoligonucleotides have been employed as therapeutic moieties in thetreatment of disease states in animals and man. Antisenseoligonucleotide drugs, including ribozymes, have been safely andeffectively administered to humans and numerous clinical trials arepresently underway. It is thus established that oligonucleotides can beuseful therapeutic modalities that can be configured to be useful intreatment regimes for treatment of cells, tissues and animals,especially humans.

[0035] In the context of this invention, the term “oligonucleotide”refers to an oligomer or polymer of ribonucleic acid (RNA) ordeoxyribonucleic acid (DNA) or mimetics thereof. This term includesoligonucleotides composed of naturally-occurring nucleobases, sugars andcovalent internucleoside (backbone) linkages as well as oligonucleotideshaving non-naturally-occurring portions which function similarly. Suchmodified or substituted oligonucleotides are often preferred over nativeforms because of desirable properties such as, for example, enhancedcellular uptake, enhanced affinity for nucleic acid target and increasedstability in the presence of nucleases.

[0036] While antisense oligonucleotides are a preferred form ofantisense compound, the present invention comprehends other oligomericantisense compounds, including but not limited to oligonucleotidemimetics such as are described below. The antisense compounds inaccordance with this invention preferably comprise from about 8 to about80 nucleobases (i.e. from about 8 to about 80 linked nucleosides).Particularly preferred antisense compounds are antisenseoligonucleotides from about 8 to about 50 nucleobases, even morepreferably those comprising from about 12 to about 30 nucleobases.Antisense compounds include ribozymes, external guide sequence (EGS)oligonucleotides (oligozymes), and other short catalytic RNAs orcatalytic oligonucleotides which hybridize to the target nucleic acidand modulate its expression.

[0037] Antisense compounds 8-80 nucleobases in length comprising astretch of at least eight (8) consecutive nucleobases selected fromwithin the illustrative antisense compounds are considered to besuitable antisense compounds as well.

[0038] Exemplary preferred antisense compounds include DNA or RNAsequences that comprise at least the 8 consecutive nucleobases from the5′-terminus of one of the illustrative preferred antisense compounds(the remaining nucleobases being a consecutive stretch of the same DNAor RNA beginning immediately upstream of the 5′-terminus of theantisense compound which is specifically hybridizable to the targetnucleic acid and continuing until the DNA or RNA contains about 8 toabout 80 nucleobases). Similarly preferred antisense compounds arerepresented by DNA or RNA sequences that comprise at least the 8consecutive nucleobases from the 3′-terminus of one of the illustrativepreferred antisense compounds (the remaining nucleobases being aconsecutive stretch of the same DNA or RNA beginning immediatelydownstream of the 3′-terminus of the antisense compound which isspecifically hybridizable to the target nucleic acid and continuinguntil the DNA or RNA contains about 8 to about 80 nucleobases). Onehaving skill in the art, once armed with the empirically-derivedpreferred antisense compounds illustrated herein will be able, withoutundue experimentation, to identify further preferred antisensecompounds.

[0039] Antisense and other compounds of the invention, which hybridizeto the target and inhibit expression of the target, are identifiedthrough experimentation, and representative sequences of these compoundsare herein identified as preferred embodiments of the invention. Whilespecific sequences of the antisense compounds are set forth herein, oneof skill in the art will recognize that these serve to illustrate anddescribe particular embodiments within the scope of the presentinvention. Additional preferred antisense compounds may be identified byone having ordinary skill.

[0040] As is known in the art, a nucleoside is a base-sugar combination.The base portion of the nucleoside is normally a heterocyclic base. Thetwo most common classes of such heterocyclic bases are the purines andthe pyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxylmoiety of the sugar. In forming oligonucleotides, the phosphate groupscovalently link adjacent nucleosides to one another to form a linearpolymeric compound. In turn, the respective ends of this linearpolymeric structure can be further joined to form a circular structure,however, open linear structures are generally preferred. In addition,linear structures may also have internal nucleobase complementarity andmay therefore fold in a manner as to produce a double strandedstructure. Within the oligonucleotide structure, the phosphate groupsare commonly referred to as forming the internucleoside backbone of theoligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′to 5′ phosphodiester linkage.

[0041] Specific examples of preferred antisense compounds useful in thisinvention include oligonucleotides containing modified backbones ornon-natural internucleoside linkages. As defined in this specification,oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone and those that do not have a phosphorusatom in the backbone. For the purposes of this specification, and assometimes referenced in the art, modified oligonucleotides that do nothave a phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

[0042] Preferred modified oligonucleotide backbones include, forexample, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotri-esters,methyl and other alkyl phosphonates including 3′-alkylene phosphonates,5′-alkylene phosphonates and chiral phosphonates, phosphinates,phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphatesand borano-phosphates having normal 3′-5′ linkages, 2′-5′ linked analogsof these, and those having inverted polarity wherein one or moreinternucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage.Preferred oligonucleotides having inverted polarity comprise a single 3′to 3′ linkage at the 3′-most internucleotide linkage i.e. a singleinverted nucleoside residue which may be abasic (the nucleobase ismissing or has a hydroxyl group in place thereof). Various salts, mixedsalts and free acid forms are also included.

[0043] Representative U.S. patents that teach the preparation of theabove phosphorus-containing linkages include, but are not limited to,U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and5,625,050, certain of which are commonly owned with this application,and each of which is herein incorporated by reference.

[0044] Preferred modified oligonucleotide backbones that do not includea phosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; riboacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH₂ component parts.

[0045] Representative U.S. patents that teach the preparation of theabove oligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain ofwhich are commonly owned with this application, and each of which isherein incorporated by reference.

[0046] In other preferred oligonucleotide mimetics, both the sugar andthe internucleoside linkage, i.e., the backbone, of the nucleotide unitsare replaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an oligonucleotide mimetic that has been shown tohave excellent hybridization properties, is referred to as a peptidenucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative U.S. patents that teach thepreparation of PNA compounds include, but are not limited to, U.S. Pat.Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al., Science, 1991, 254, 1497-1500.

[0047] Most preferred embodiments of the invention are oligonucleotideswith phosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [knownas a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃) —N(CH₃) —CH₂— and —O—N(CH₃)—CH₂—CH₂— [wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of the abovereferenced U.S. Pat. No. 5,489,677, and the amide backbones of the abovereferenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotideshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

[0048] Modified oligonucleotides may also contain one or moresubstituted sugar moieties. Preferred oligonucleotides comprise one ofthe following at the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, orN-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkylor C₂ to C₁₀ alkenyl and alkynyl. Particularly preferred areO[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃,O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃]₂, where n and m are from1 to about 10. Other preferred oligonucleotides comprise one of thefollowing at the 2′ position: C₁ to C₁₀ lower alkyl, substituted loweralkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH,SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of anoligonucleotide, or a group for improving the pharmacodynamic propertiesof an oligonucleotide, and other substituents having similar properties.A preferred modification includes 2′-methoxyethoxy (2′—O—CH₂CH₂OCH₃,also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv.Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A furtherpreferred modification includes 2′-dimethylaminooxyethoxy, i.e., aO(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in exampleshereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e.,2′—O—CH₂—O—CH₂—N(CH₃)₂, also described in examples hereinbelow.

[0049] Other preferred modifications include 2′-methoxy (2′—O—CH₃),2′-aminopropoxy (2′—OCH₂CH₂CH₂NH₂), 2′-allyl (2′—CH₂—CH═CH₂), 2′-O-allyl(2′—O—CH₂—CH═CH₂) and 2′-fluoro (2′—F). The 2′-modification may be inthe arabino (up) position or ribo (down) position. A preferred2′-arabino modification is 2′—F. Similar modifications may also be madeat other positions on the oligonucleotide, particularly the 3′ positionof the sugar on the 3′ terminal nucleotide or in 2′-5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotide.Oligonucleotides may also have sugar mimetics such as cyclobutylmoieties in place of the pentofuranosyl sugar. Representative U.S.patents that teach the preparation of such modified sugar structuresinclude, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800;5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785;5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300;5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; and5,700,920, certain of which are commonly owned with the instantapplication, and each of which is herein incorporated by reference inits entirety.

[0050] A further preferred modification includes Locked Nucleic Acids(LNAs) in which the 2′-hydroxyl group is linked to the 3′ or 4′ carbonatom of the sugar ring thereby forming a bicyclic sugar moiety. Thelinkage is preferably a methelyne (—CH₂—)_(n) group bridging the 2′oxygen atom and the 4′ carbon atom wherein n is 1 or 2. LNAs andpreparation thereof are described in WO 98/39352 and WO 99/14226.

[0051] Oligonucleotides may also include nucleobase (often referred toin the art simply as “base”) modifications or substitutions. As usedherein, “unmodified” or “natural” nucleobases include the purine basesadenine (A) and guanine (G), and the pyrimidine bases thymine (T),cytosine (C) and uracil (U). Modified nucleobases include othersynthetic and natural nucleobases such as 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl(—C≡C—CH₃) uracil and cytosine and other alkynyl derivatives ofpyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl and other 8-substituted adenines and guanines, 5-haloparticularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine,2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modifiednucleobases include tricyclic pyrimidines such as phenoxazinecytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazinecytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps suchas a substituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine(H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobasesmay also include those in which the purine or pyrimidine base isreplaced with other heterocycles, for example 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobasesinclude those disclosed in U.S. Pat. No. 3,687,808, those disclosed inThe Concise Encyclopedia Of Polymer Science And Engineering, pages858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosedby Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and those disclosed by Sanghvi, Y. S., Chapter 15, AntisenseResearch and Applications, pages 289-302, Crooke, S. T. and Lebleu, B.ed., CRC Press, 1993. Certain of these nucleobases are particularlyuseful for increasing the binding affinity of the oligomeric compoundsof the invention. These include 5-substituted pyrimidines,6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. andLebleu, B., eds., Antisense Research and Applications, CRC Press, BocaRaton, 1993, pp. 276-278) and are presently preferred basesubstitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications.

[0052] Representative U.S. patents that teach the preparation of certainof the above noted modified nucleobases as well as other modifiednucleobases include, but are not limited to, the above noted U.S. Pat.No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121,5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; and5,681,941, certain of which are commonly owned with the instantapplication, and each of which is herein incorporated by reference, andU.S. Pat. No. 5,750,692, which is commonly owned with the instantapplication and also herein incorporated by reference.

[0053] Another modification of the oligonucleotides of the inventioninvolves chemically linking to the oligonucleotide one or more moietiesor conjugates which enhance the activity, cellular distribution orcellular uptake of the oligonucleotide. The compounds of the inventioncan include conjugate groups covalently bound to functional groups suchas primary or secondary hydroxyl groups. Conjugate groups of theinvention include intercalators, reporter molecules, polyamines,polyamides, polyethylene glycols, polyethers, groups that enhance thepharmacodynamic properties of oligomers, and groups that enhance thepharmacokinetic properties of oligomers. Typical conjugate groupsinclude cholesterols, lipids, phospholipids, biotin, phenazine, folate,phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines,coumarins, and dyes. Groups that enhance the pharmacodynamic properties,in the context of this invention, include groups that improve oligomeruptake, enhance oligomer resistance to degradation, and/or strengthensequence-specific hybridization with RNA. Groups that enhance thepharmacokinetic properties, in the context of this invention, includegroups that improve oligomer uptake, distribution, metabolism orexcretion. Representative conjugate groups are disclosed inInternational Patent Application PCT/US92/09196, filed Oct. 23, 1992 theentire disclosure of which is incorporated herein by reference.Conjugate moieties include but are not limited to lipid moieties such asa cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA,1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem.Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharanet al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphaticchain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al.,EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259,327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid,e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937). Oligonucleotides of the invention mayalso be conjugated to active drug substances, for example, aspirin,warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen,(S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoicacid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide,a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug,an antidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drugconjugates and their preparation are described in U.S. patentapplication Ser. No. 09/334,130 (filed Jun. 15, 1999) which isincorporated herein by reference in its entirety.

[0054] Representative U.S. patents that teach the preparation of sucholigonucleotide conjugates include, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, certain ofwhich are commonly owned with the instant application, and each of whichis herein incorporated by reference.

[0055] It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligonucleotide. The present invention alsoincludes antisense compounds which are chimeric compounds. “Chimeric”antisense compounds or “chimeras,” in the context of this invention, areantisense compounds, particularly oligonucleotides, which contain two ormore chemically distinct regions, each made up of at least one monomerunit, i.e., a nucleotide in the case of an oligonucleotide compound.These oligonucleotides typically contain at least one region wherein theoligonucleotide is modified so as to confer upon the oligonucleotideincreased resistance to nuclease degradation, increased cellular uptake,increased stability and/or increased binding affinity for the targetnucleic acid. An additional region of the oligonucleotide may serve as asubstrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. Byway of example, RNAse H is a cellular endonuclease which cleaves the RNAstrand of an RNA:DNA duplex. Activation of RNase H, therefore, resultsin cleavage of the RNA target, thereby greatly enhancing the efficiencyof oligonucleotide inhibition of gene expression. The cleavage ofRNA:RNA hybrids can, in like fashion, be accomplished through theactions of endoribonucleases, such as interferon-induced RNAseL whichcleaves both cellular and viral RNA. Consequently, comparable resultscan often be obtained with shorter oligonucleotides when chimericoligonucleotides are used, compared to phosphorothioatedeoxyoligonucleotides hybridizing to the same target region. Cleavage ofthe RNA target can be routinely detected by gel electrophoresis and, ifnecessary, associated nucleic acid hybridization techniques known in theart.

[0056] Chimeric antisense compounds of the invention may be formed ascomposite structures of two or more oligonucleotides, modifiedoligonucleotides, oligonucleosides and/or oligonucleotide mimetics asdescribed above. Such compounds have also been referred to in the art ashybrids or gapmers. Representative U.S. patents that teach thepreparation of such hybrid structures include, but are not limited to,U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878;5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and5,700,922, certain of which are commonly owned with the instantapplication, and each of which is herein incorporated by reference inits entirety.

[0057] The antisense compounds used in accordance with this inventionmay be conveniently and routinely made through the well-known techniqueof solid phase synthesis. Equipment for such synthesis is sold byseveral vendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and alkylated derivatives.

[0058] The compounds of the invention may also be admixed, encapsulated,conjugated or otherwise associated with other molecules, moleculestructures or mixtures of compounds, as for example, liposomes,receptor-targeted molecules, oral, rectal, topical or otherformulations, for assisting in uptake, distribution and/or absorption.Representative U.S. patents that teach the preparation of such uptake,distribution and/or absorption-assisting formulations include, but arenot limited to, U.S. Pat. Nos. 5,108,921; 5;354,844; 5,416,016;5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;5,580,575; and 5,595,756, each of which is herein incorporated byreference.

[0059] The antisense compounds of the invention encompass anypharmaceutically acceptable salts, esters, or salts of such esters, orany other compound which, upon administration to an animal, including ahuman, is capable of providing (directly or indirectly) the biologicallyactive metabolite or residue thereof. Accordingly, for example, thedisclosure is also drawn to prodrugs and pharmaceutically acceptablesalts of the compounds of the invention, pharmaceutically acceptablesalts of such prodrugs, and other bioequivalents.

[0060] The term “prodrug” indicates a therapeutic agent that is preparedin an inactive form that is converted to an active form (i.e., drug)within the body or cells thereof by the action of endogenous enzymes orother chemicals and/or conditions. In particular, prodrug versions ofthe oligonucleotides of the invention are prepared as SATE[(S-acetyl-2-thioethyl) phosphate] derivatives according to the methodsdisclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 orin WO 94/26764 and U.S. Pat. No. 5,770,713 to Imbach et al.

[0061] The term “pharmaceutically acceptable salts” refers tophysiologically and pharmaceutically acceptable salts of the compoundsof the invention: i.e., salts that retain the desired biologicalactivity of the parent compound and do not impart undesiredtoxicological effects thereto.

[0062] Pharmaceutically acceptable base addition salts are formed withmetals or amines, such as alkali and alkaline earth metals or organicamines. Examples of metals used as cations are sodium, potassium,magnesium, calcium, and the like. Examples of suitable amines areN,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine(see, for example, Berge et al., “Pharmaceutical Salts,” J. of PharmaSci., 1977, 66, 1-19). The base addition salts of said acidic compoundsare prepared by contacting the free acid form with a sufficient amountof the desired base to produce the salt in the conventional manner. Thefree acid form may be regenerated by contacting the salt form with anacid and isolating the free acid in the conventional manner. The freeacid forms differ from their respective salt forms somewhat in certainphysical properties such as solubility in polar solvents, but otherwisethe salts are equivalent to their respective free acid for purposes ofthe present invention. As used herein, a “pharmaceutical addition salt”includes a pharmaceutically acceptable salt of an acid form of one ofthe components of the compositions of the invention. These includeorganic or inorganic acid salts of the amines. Preferred acid salts arethe hydrochlorides, acetates, salicylates, nitrates and phosphates.Other suitable pharmaceutically acceptable salts are well known to thoseskilled in the art and include basic salts of a variety of inorganic andorganic acids, such as, for example, with inorganic acids, such as forexample hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoricacid; with organic carboxylic, sulfonic, sulfo or phospho acids orN-substituted sulfamic acids, for example acetic acid, propionic acid,glycolic acid, succinic acid, maleic acid, hydroxymaleic acid,methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid,oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, salicylic acid,4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid,embonic acid, nicotinic acid or isonicotinic acid; and with amino acids,such as the 20 alpha-amino acids involved in the synthesis of proteinsin nature, for example glutamic acid or aspartic acid, and also withphenylacetic acid, methanesulfonic acid, ethanesulfonic acid,2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid,benzenesulfonic acid, 4-methylbenzenesulfonic acid,naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (withthe formation of cyclamates), or with other acid organic compounds, suchas ascorbic acid. Pharmaceutically acceptable salts of compounds mayalso be prepared with a pharmaceutically acceptable cation. Suitablepharmaceutically acceptable cations are well known to those skilled inthe art and include alkaline, alkaline earth, ammonium and quaternaryammonium cations. Carbonates or hydrogen carbonates are also possible.

[0063] For oligonucleotides, preferred examples of pharmaceuticallyacceptable salts include but are not limited to (a) salts formed withcations such as sodium, potassium, ammonium, magnesium, calcium,polyamines such as spermine and spermidine, etc.; (b) acid additionsalts formed with inorganic acids, for example hydrochloric acid,hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and thelike; (c) salts formed with organic acids such as, for example, aceticacid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaricacid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoicacid, tannic acid, palmitic acid, alginic acid, polyglutamic acid,naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d)salts formed from elemental anions such as chlorine, bromine, andiodine.

[0064] The antisense compounds of the present invention can be utilizedfor diagnostics, therapeutics, prophylaxis and as research reagents andkits. For therapeutics, an animal, preferably a human, suspected ofhaving a disease or disorder which can be treated by modulating theexpression of resistin is treated by administering antisense compoundsin accordance with this invention. The compounds of the invention can beutilized in pharmaceutical compositions by adding an effective amount ofan antisense compound to a suitable pharmaceutically acceptable diluentor carrier. Use of the antisense compounds and methods of the inventionmay also be useful prophylactically, e.g., to prevent or delayinfection, inflammation or tumor formation, for example.

[0065] The antisense compounds of the invention are useful for researchand diagnostics, because these compounds hybridize to nucleic acidsencoding resistin, enabling sandwich and other assays to easily beconstructed to exploit this fact. Hybridization of the antisenseoligonucleotides of the invention with a nucleic acid encoding resistincan be detected by means known in the art. Such means may includeconjugation of an enzyme to the oligonucleotide, radiolabelling of theoligonucleotide or any other suitable detection means. Kits using suchdetection means for detecting the level of resistin in a sample may alsobe prepared.

[0066] The present invention also includes pharmaceutical compositionsand formulations which include the antisense compounds of the invention.The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic and to mucousmembranes including vaginal and rectal delivery), pulmonary, e.g., byinhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal), oralor parenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; or intracranial, e.g., intrathecal or intraventricular,administration. Oligonucleotides with at least one 2′-O-methoxyethylmodification are believed to be particularly useful for oraladministration.

[0067] Pharmaceutical compositions and formulations for topicaladministration may include transdermal patches, ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable. Coated condoms,gloves and the like may also be useful. Preferred topical formulationsinclude those in which the oligonucleotides of the invention are inadmixture with a topical delivery agent such as lipids, liposomes, fattyacids, fatty acid esters, steroids, chelating agents and surfactants.Preferred lipids and liposomes include neutral (e.g.dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl cholineDMPC, distearolyphosphatidyl choline) negative (e.g.dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g.dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA). Oligonucleotides of the invention may beencapsulated within liposomes or may form complexes thereto, inparticular to cationic liposomes. Alternatively, oligonucleotides may becomplexed to lipids, in particular to cationic lipids. Preferred fattyacids and esters include but are not limited arachidonic acid, oleicacid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristicacid, palmitic acid, stearic acid, linoleic acid, linolenic acid,dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or aC₁₋₁₀ alkyl ester (e.g. isopropylmyristate IPM), monoglyceride,diglyceride or pharmaceutically acceptable salt thereof. Topicalformulations are described in detail in U.S. patent application Ser. No.09/315,298 filed on May 20, 1999 which is incorporated herein byreference in its entirety.

[0068] Compositions and formulations for oral administration includepowders or granules, microparticulates, nanoparticulates, suspensions orsolutions in water or non-aqueous media, capsules, gel capsules,sachets, tablets or minitablets. Thickeners, flavoring agents, diluents,emulsifiers, dispersing aids or binders may be desirable. Preferred oralformulations are those in which oligonucleotides of the invention areadministered in conjunction with one or more penetration enhancerssurfactants and chelators. Preferred surfactants include fatty acidsand/or esters or salts thereof, bile acids and/or salts thereof.Preferred bile acids/salts include chenodeoxycholic acid (CDCA) andursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid,deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid,taurocholic acid, taurodeoxycholic acid, sodiumtauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Preferredfatty acids include arachidonic acid, undecanoic acid, oleic acid,lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid,stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate,monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or amonoglyceride, a diglyceride or a pharmaceutically acceptable saltthereof (e.g. sodium). Also preferred are combinations of penetrationenhancers, for example, fatty acids/salts in combination with bileacids/salts. A particularly preferred combination is the sodium salt oflauric acid, capric acid and UDCA. Further penetration enhancers includepolyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether.Oligonucleotides of the invention may be delivered orally, in granularform including sprayed dried particles, or complexed to form micro ornanoparticles. Oligonucleotide complexing agents include poly-aminoacids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes,polyalkylcyanoacrylates; cationized gelatins, albumins, starches,acrylates, polyethyleneglycols (PEG) and starches;polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans,celluloses and starches. Particularly preferred completing agentsinclude chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine,polyornithine, polyspermines, protamine, polyvinylpyridine,polythiodiethylamino-methylethylene P(TDAE), polyaminostyrene (e.g.p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate),poly(butylcyanoacrylate), poly(isobutylcyanoacrylate),poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate,DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate,polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolicacid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulationsfor oligonucleotides and their preparation are described in detail inU.S. application Ser. Nos. 08/886,829 (filed Jul. 1, 1997), 09/108,673(filed Jul. 1, 1998), 09/256,515 (filed Feb. 23, 1999), 09/082,624(filed May 21, 1998) and 09/315,298 (filed May 20, 1999), each of whichis incorporated herein by reference in their entirety.

[0069] Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionswhich may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

[0070] Pharmaceutical compositions of the present invention include, butare not limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids.

[0071] The pharmaceutical formulations of the present invention, whichmay conveniently be presented in unit dosage form, may be preparedaccording to conventional techniques well known in the pharmaceuticalindustry. Such techniques include the step of bringing into associationthe active ingredients with the pharmaceutical carrier(s) orexcipient(s). In general, the formulations are prepared by uniformly andintimately bringing into association the active ingredients with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product.

[0072] The compositions of the present invention may be formulated intoany of many possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention may also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions may further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

[0073] In one embodiment of the present invention the pharmaceuticalcompositions may be formulated and used as foams. Pharmaceutical foamsinclude formulations such as, but not limited to, emulsions,microemulsions, creams, jellies and liposomes. While basically similarin nature these formulations vary in the components and the consistencyof the final product. The preparation of such compositions andformulations is generally known to those skilled in the pharmaceuticaland formulation arts and may be applied to the formulation of thecompositions of the present invention.

[0074] Emulsions

[0075] The compositions of the present invention may be prepared andformulated as emulsions. Emulsions are typically heterogenous systems ofone liquid dispersed in another in the form of droplets usuallyexceeding 0.1 μm in diameter (Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 2, p. 335; Higuchi et al., in Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p.301). Emulsions are often biphasic systems comprising two immiscibleliquid phases intimately mixed and dispersed with each other. Ingeneral, emulsions may be of either the water-in-oil (w/o) or theoil-in-water (o/w) variety. When an aqueous phase is finely divided intoand dispersed as minute droplets into a bulk oily phase, the resultingcomposition is called a water-in-oil (w/o) emulsion. Alternatively, whenan oily phase is finely divided into and dispersed as minute dropletsinto a bulk aqueous phase, the resulting composition is called anoil-in-water (o/w) emulsion. Emulsions may contain additional componentsin addition to the dispersed phases, and the active drug which may bepresent as a solution in either the aqueous phase, oily phase or itselfas a separate phase. Pharmaceutical excipients such as emulsifiers,stabilizers, dyes, and anti-oxidants may also be present in emulsions asneeded. Pharmaceutical emulsions may also be multiple emulsions that arecomprised of more than two phases such as, for example, in the case ofoil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions.Such complex formulations often provide certain advantages that simplebinary emulsions do not. Multiple emulsions in which individual oildroplets of an o/w emulsion enclose small water droplets constitute aw/o/w emulsion. Likewise a system of oil droplets enclosed in globulesof water stabilized in an oily continuous phase provides an o/w/oemulsion.

[0076] Emulsions are characterized by little or no thermodynamicstability. Often, the dispersed or discontinuous phase of the emulsionis well dispersed into the external or continuous phase and maintainedin this form through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion may be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatmay be incorporated into either phase of the emulsion. Emulsifiers maybroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199).

[0077] Synthetic surfactants, also known as surface active agents, havefound wide applicability in the formulation of emulsions and have beenreviewed in the literature (Rieger, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York,N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic andcomprise a hydrophilic and a hydrophobic portion. The ratio of thehydrophilic to the hydrophobic nature of the surfactant has been termedthe hydrophile/lipophile balance (HLB) and is a valuable tool incategorizing and selecting surfactants in the preparation offormulations. Surfactants may be classified into different classes basedon the nature of the hydrophilic group: nonionic, anionic, cationic andamphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Riegerand Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,p. 285).

[0078] Naturally occurring emulsifiers used in emulsion formulationsinclude lanolin, beeswax, phosphatides, lecithin and acacia. Absorptionbases possess hydrophilic properties such that they can soak up water toform w/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

[0079] A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

[0080] Hydrophilic colloids or hydrocolloids include naturally occurringgums and synthetic polymers such as polysaccharides (for example,acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, andtragacanth), cellulose derivatives (for example, carboxymethylcelluloseand carboxypropylcellulose), and synthetic polymers (for example,carbomers, cellulose ethers, and carboxyvinyl polymers). These disperseor swell in water to form colloidal solutions that stabilize emulsionsby forming strong interfacial films around the dispersed-phase dropletsand by increasing the viscosity of the external phase.

[0081] Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that may readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used may be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

[0082] The application of emulsion formulations via dermatological, oraland parenteral routes and methods for their manufacture have beenreviewed in the literature (Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 199). Emulsion formulations for oral deliveryhave been very widely used because of ease of formulation, as well asefficacy from an absorption and bioavailability standpoint (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil baselaxatives, oil-soluble vitamins and high fat nutritive preparations areamong the materials that have commonly been administered orally as o/wemulsions.

[0083] In one embodiment of the present invention, the compositions ofoligonucleotides and nucleic acids are formulated as microemulsions. Amicroemulsion may be defined as a system of water, oil and amphiphilewhich is a single optically isotropic and thermodynamically stableliquid solution (Rosoff, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 245). Typically microemulsions are systems that areprepared by first dispersing an oil in an aqueous surfactant solutionand then adding a sufficient amount of a fourth component, generally anintermediate chain-length alcohol to form a transparent system.Therefore, microemulsions have also been described as thermodynamicallystable, isotropically clear dispersions of two immiscible liquids thatare stabilized by interfacial films of surface-active molecules (Leungand Shah, in: Controlled Release of Drugs: Polymers and AggregateSystems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages185-215). Microemulsions commonly are prepared via a combination ofthree to five components that include oil, water, surfactant,cosurfactant and electrolyte. Whether the microemulsion is of thewater-in-oil (w/o) or an oil-in-water (o/w) type is dependent on theproperties of the oil and surfactant used and on the structure andgeometric packing of the polar heads and hydrocarbon tails of thesurfactant molecules (Schott, in Remington's Pharmaceutical Sciences,Mack Publishing Co., Easton, Pa., 1985, p. 271).

[0084] The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared toconventional emulsions, microemulsions offer the advantage ofsolubilizing water-insoluble drugs in a formulation of thermodynamicallystable droplets that are formed spontaneously.

[0085] Surfactants used in the preparation of microemulsions include,but are not limited to, ionic surfactants, non-ionic surfactants, Brij96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions may, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase may typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase may include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C8-C12) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils and silicone oil.

[0086] Microemulsions are particularly of interest from the standpointof drug solubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (Constantinideset al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm.Sci., 1996, 85, 138-143). Often microemulsions may form spontaneouslywhen their components are brought together at ambient temperature. Thismay be particularly advantageous when formulating thermolabile drugs,peptides or oligonucleotides. Microemulsions have also been effective inthe transdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of oligonucleotides and nucleic acidsfrom the gastrointestinal tract, as well as improve the local cellularuptake of oligonucleotides and nucleic acids within the gastrointestinaltract, vagina, buccal cavity and other areas of administration.

[0087] Microemulsions of the present invention may also containadditional components and additives such as sorbitan monostearate (Grill3), Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the oligonucleotides andnucleic acids of the present invention. Penetration enhancers used inthe microemulsions of the present invention may be classified asbelonging to one of five broad categories—surfactants, fatty acids, bilesalts, chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Eachof these classes has been discussed above.

[0088] Liposomes

[0089] There are many organized surfactant structures besidesmicroemulsions that have been studied and used for the formulation ofdrugs. These include monolayers, micelles, bilayers and vesicles.Vesicles, such as liposomes, have attracted great interest because oftheir specificity and the duration of action they offer from thestandpoint of drug delivery. As used in the present invention, the term“liposome” means a vesicle composed of amphiphilic lipids arranged in aspherical bilayer or bilayers.

[0090] Liposomes are unilamellar or multilamellar vesicles which have amembrane formed from a lipophilic material and an aqueous interior. Theaqueous portion contains the composition to be delivered. Cationicliposomes possess the advantage of being able to fuse to the cell wall.Non-cationic liposomes, although not able to fuse as efficiently withthe cell wall, are taken up by macrophages in vivo.

[0091] In order to cross intact mammalian skin, lipid vesicles must passthrough a series of fine pores, each with a diameter less than 50 nm,under the influence of a suitable transdermal gradient. Therefore, it isdesirable to use a liposome which is highly deformable and able to passthrough such fine pores.

[0092] Further advantages of liposomes include; liposomes obtained fromnatural phospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated drugs in their internal compartments frommetabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245). Important considerations in thepreparation of liposome formulations are the lipid surface charge,vesicle size and the aqueous volume of the liposomes.

[0093] Liposomes are useful for the transfer and delivery of activeingredients to the site of action. Because the liposomal membrane isstructurally similar to biological membranes, when liposomes are appliedto a tissue, the liposomes start to merge with the cellular membranesand as the merging of the liposome and cell progresses, the liposomalcontents are emptied into the cell where the active agent may act.

[0094] Liposomal formulations have been the focus of extensiveinvestigation as the mode of delivery for many drugs. There is growingevidence that for topical administration, liposomes present severaladvantages over other formulations. Such advantages include reducedside-effects related to high systemic absorption of the administereddrug, increased accumulation of the administered drug at the desiredtarget, and the ability to administer a wide variety of drugs, bothhydrophilic and hydrophobic, into the skin.

[0095] Several reports have detailed the ability of liposomes to deliveragents including high-molecular weight DNA into the skin. Compoundsincluding analgesics, antibodies, hormones and high-molecular weightDNAs have been administered to the skin. The majority of applicationsresulted in the targeting of the upper epidermis.

[0096] Liposomes fall into two broad classes. Cationic liposomes arepositively charged liposomes which interact with the negatively chargedDNA molecules to form a stable complex. The positively chargedDNA/liposome complex binds to the negatively charged cell surface and isinternalized in an endosome. Due to the acidic pH within the endosome,the liposomes are ruptured, releasing their contents into the cellcytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147,980-985).

[0097] Liposomes which are pH-sensitive or negatively-charged, entrapDNA rather than complex with it. Since both the DNA and the lipid aresimilarly charged, repulsion rather than complex formation occurs.Nevertheless, some DNA is entrapped within the aqueous interior of theseliposomes. pH-sensitive liposomes have been used to deliver DNA encodingthe thymidine kinase gene to cell monolayers in culture. Expression ofthe exogenous gene was detected in the target cells (Zhou et al.,Journal of Controlled Release, 1992, 19, 269-274).

[0098] One major type of liposomal composition includes phospholipidsother than naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

[0099] Several studies have assessed the topical delivery of liposomaldrug formulations to the skin. Application of liposomes containinginterferon to guinea pig skin resulted in a reduction of skin herpessores while delivery of interferon via other means (e.g. as a solutionor as an emulsion) were ineffective (Weiner et al., Journal of DrugTargeting, 1992, 2, 405-410). Further, an additional study tested theefficacy of interferon administered as part of a liposomal formulationto the administration of interferon using an aqueous system, andconcluded that the liposomal formulation was superior to aqueousadministration (du Plessis et al., Antiviral Research, 1992, 18,259-265).

[0100] Non-ionic liposomal systems have also been examined to determinetheir utility in the delivery of drugs to the skin, in particularsystems comprising non-ionic surfactant and cholesterol. Non-ionicliposomal formulations comprising Novasome™ I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver cyclosporin-A into the dermis of mouse skin. Resultsindicated that such non-ionic liposomal systems were effective infacilitating the deposition of cyclosporin-A into different layers ofthe skin (Hu et al. S.T.P. Pharma. Sci., 1994, 4, 6, 466).

[0101] Liposomes also include “sterically stabilized” liposomes, a termwhich, as used herein, refers to liposomes comprising one or morespecialized lipids that, when incorporated into liposomes, result inenhanced circulation lifetimes relative to liposomes lacking suchspecialized lipids. Examples of sterically stabilized liposomes arethose in which part of the vesicle-forming lipid portion of the liposome(A) comprises one or more glycolipids, such as monosialogangliosideG_(M1), or (B) is derivatized with one or more hydrophilic polymers,such as a polyethylene glycol (PEG) moiety. While not wishing to bebound by any particular theory, it is thought in the art that, at leastfor sterically stabilized liposomes containing gangliosides,sphingomyelin, or PEG-derivatized lipids, the enhanced circulationhalf-life of these sterically stabilized liposomes derives from areduced uptake into cells of the reticuloendothelial system (RES) (Allenet al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993,53, 3765).

[0102] Various liposomes comprising one or more glycolipids are known inthe art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64)reported the ability of monosialoganglioside G_(M1), galactocerebrosidesulfate and phosphatidylinositol to improve blood half-lives ofliposomes. These findings were expounded upon by Gabizon et al. (Proc.Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO88/04924, both to Allen et al., disclose liposomes comprising (1)sphingomyelin and (2) the ganglioside G_(M1), or a galactocerebrosidesulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomescomprising sphingomyelin. Liposomes comprising1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Limet al.).

[0103] Many liposomes comprising lipids derivatized with one or morehydrophilic polymers, and methods of preparation thereof, are known inthe art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778)described liposomes comprising a nonionic detergent, 2C₁₂15G, thatcontains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) notedthat hydrophilic coating of polystyrene particles with polymeric glycolsresults in significantly enhanced blood half-lives. Syntheticphospholipids modified by the attachment of carboxylic groups ofpolyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos.4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235)described experiments demonstrating that liposomes comprisingphosphatidylethanolamine (PE) derivatized with PEG or PEG stearate havesignificant increases in blood circulation half-lives. Blume et al.(Biochimica et Biophysica Acta, 1990, 1029, 91) extended suchobservations to other PEG-derivatized phospholipids, e.g., DSPE-PEG,formed from the combination of distearoylphosphatidylethanolamine (DSPE)and PEG. Liposomes having covalently bound PEG moieties on theirexternal surface are described in European Patent No. EP 0 445 131 B1and WO 90/04384 to Fisher. Liposome compositions containing 1-20 molepercent of PE derivatized with PEG, and methods of use thereof, aredescribed by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) andMartin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496813 B1). Liposomes comprising a number of other lipid-polymer conjugatesare disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martinet al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprisingPEG-modified ceramide lipids are described in WO 96/10391 (Choi et al.).U.S. Pat. Nos. 5,540,935 (Miyazaki et al.) and 5,556,948 (Tagawa et al.)describe PEG-containing liposomes that can be further derivatized withfunctional moieties on their surfaces.

[0104] A limited number of liposomes comprising nucleic acids are knownin the art. WO 96/40062 to Thierry et al. discloses methods forencapsulating high molecular weight nucleic acids in liposomes. U.S.Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomesand asserts that the contents of such liposomes may include an antisenseRNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methodsof encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Loveet al. discloses liposomes comprising antisense oligonucleotidestargeted to the raf gene.

[0105] Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes may be described as lipid dropletswhich are so highly deformable that they are easily able to penetratethrough pores which are smaller than the droplet. Transfersomes areadaptable to the environment in which they are used, e.g. they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. To make transfersomes it is possible to add surfaceedge-activators, usually surfactants, to a standard liposomalcomposition. Transfersomes have been used to deliver serum albumin tothe skin. The transfersome-mediated delivery of serum albumin has beenshown to be as effective as subcutaneous injection of a solutioncontaining serum albumin.

[0106] Surfactants find wide application in formulations such asemulsions (including microemulsions) and liposomes. The most common wayof classifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, inPharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988,p. 285).

[0107] If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

[0108] If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

[0109] If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

[0110] If the surfactant molecule has the ability to carry either apositive or negative charge, the surfactant is classified as amphoteric.Amphoteric surfactants include acrylic acid derivatives, substitutedalkylamides, N-alkylbetaines and phosphatides.

[0111] The use of surfactants in drug products, formulations and inemulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms,Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

[0112] Penetration Enhancers

[0113] In one embodiment, the present invention employs variouspenetration enhancers to effect the efficient delivery of nucleic acids,particularly oligonucleotides, to the skin of animals. Most drugs arepresent in solution in both ionized and nonionized forms. However,usually only lipid soluble or lipophilic drugs readily cross cellmembranes. It has been discovered that even non-lipophilic drugs maycross cell membranes if the membrane to be crossed is treated with apenetration enhancer. In addition to aiding the diffusion ofnon-lipophilic drugs across cell membranes, penetration enhancers alsoenhance the permeability of lipophilic drugs.

[0114] Penetration enhancers may be classified as belonging to one offive broad categories, i.e., surfactants, fatty acids, bile salts,chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92). Eachof the above mentioned classes of penetration enhancers are describedbelow in greater detail.

[0115] Surfactants:

[0116] In connection with the present invention, surfactants (or“surface-active agents”) are chemical entities which, when dissolved inan aqueous solution, reduce the surface tension of the solution or theinterfacial tension between the aqueous solution and another liquid,with the result that absorption of oligonucleotides through the mucosais enhanced. In addition to bile salts and fatty acids, thesepenetration enhancers include, for example, sodium lauryl sulfate,polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991,p.92); and perfluorochemical emulsions, such as FC-43. Takahashi et al.,J. Pharm. Pharmacol., 1988, 40, 252).

[0117] Fatty Acids:

[0118] Various fatty acids and their derivatives which act aspenetration enhancers include, for example, oleic acid, lauric acid,capric acid (n-decanoic acid), myristic acid, palmitic acid, stearicacid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, C₁₋₁₀ alkyl esters thereof (e.g., methyl, isopropyl andt-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate,caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92;Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990,7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

[0119] Bile Salts:

[0120] The physiological role of bile includes the facilitation ofdispersion and absorption of lipids and fat-soluble vitamins (Brunton,Chapter 38 in: Goodman & Gilman's The Pharmacological Basis ofTherapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996,pp. 934-935). Various natural bile salts, and their syntheticderivatives, act as penetration enhancers. Thus the term “bile salts”includes any of the naturally occurring components of bile as well asany of their synthetic derivatives. The bile salts of the inventioninclude, for example, cholic acid (or its pharmaceutically acceptablesodium salt, sodium cholate), dehydrocholic acid (sodiumdehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid(sodium glucholate), glycholic acid (sodium glycocholate),glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid(sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate),chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid(UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodiumglycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee etal., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18thEd., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages782-783; Muranishi, Critical Reviews in Therapeutic Drug CarrierSystems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992,263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

[0121] Chelating Agents:

[0122] Chelating agents, as used in connection with the presentinvention, can be defined as compounds that remove metallic ions fromsolution by forming complexes therewith, with the result that absorptionof oligonucleotides through the mucosa is enhanced. With regards totheir use as penetration enhancers in the present invention, chelatingagents have the added advantage of also serving as DNase inhibitors, asmost characterized DNA nucleases require a divalent metal ion forcatalysis and are thus inhibited by chelating agents (Jarrett, J.Chromatogr., 1993, 618, 315-339). Chelating agents of the inventioninclude but are not limited to disodium ethylenediaminetetraacetate(EDTA), citric acid, salicylates (e.g., sodium salicylate,5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen,laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems,1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).

[0123] Non-Chelating Non-Surfactants:

[0124] As used herein, non-chelating non-surfactant penetrationenhancing compounds can be defined as compounds that demonstrateinsignificant activity as chelating agents or as surfactants but thatnonetheless enhance absorption of oligonucleotides through thealimentary mucosa (Muranishi, Critical Reviews in Therapeutic DrugCarrier Systems, 1990, 7, 1-33). This class of penetration enhancersinclude, for example, unsaturated cyclic ureas, 1-alkyl- and1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, page 92); and non-steroidalanti-inflammatory agents such as diclofenac sodium, indomethacin andphenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39,621-626).

[0125] Agents that enhance uptake of oligonucleotides at the cellularlevel may also be added to the pharmaceutical and other compositions ofthe present invention. For example, cationic lipids, such as lipofectin(Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives,and polycationic molecules, such as polylysine (Lollo et al., PCTApplication WO 97/30731), are also known to enhance the cellular uptakeof oligonucleotides.

[0126] Other agents may be utilized to enhance the penetration of theadministered nucleic acids, including glycols such as ethylene glycoland propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenessuch as limonene and menthone.

[0127] Carriers

[0128] Certain compositions of the present invention also incorporatecarrier compounds in the formulation. As used herein, “carrier compound”or “carrier” can refer to a nucleic acid, or analog thereof, which isinert (i.e., does not possess biological activity per se) but isrecognized as a nucleic acid by in vivo processes that reduce thebioavailability of a nucleic acid having biological activity by, forexample, degrading the biologically active nucleic acid or promoting itsremoval from circulation. The coadministration of a nucleic acid and acarrier compound, typically with an excess of the latter substance, canresult in a substantial reduction of the amount of nucleic acidrecovered in the liver, kidney or other extracirculatory reservoirs,presumably due to competition between the carrier compound and thenucleic acid for a common receptor. For example, the recovery of apartially phosphorothioate oligonucleotide in hepatic tissue can bereduced when it is coadministered with polyinosinic acid, dextransulfate, polycytidic acid or4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al.,Antisense Res. Dev., 1995, 5, 115-121; Takakura et al., Antisense &Nucl. Acid Drug Dev., 1996, 6, 177-183).

[0129] Excipients

[0130] In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient may be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutical carriers include, but are notlimited to, binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodiumstarch glycolate, etc.); and wetting agents (e.g., sodium laurylsulphate, etc.).

[0131] Pharmaceutically acceptable organic or inorganic excipientsuitable for non-parenteral administration which do not deleteriouslyreact with nucleic acids can also be used to formulate the compositionsof the present invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

[0132] Formulations for topical administration of nucleic acids mayinclude sterile and non-sterile aqueous solutions, non-aqueous solutionsin common solvents such as alcohols, or solutions of the nucleic acidsin liquid or solid oil bases. The solutions may also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration which do not deleteriously react with nucleic acids canbe used.

[0133] Suitable pharmaceutically acceptable excipients include, but arenot limited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike.

[0134] Other Components

[0135] The compositions of the present invention may additionallycontain other adjunct components conventionally found in pharmaceuticalcompositions, at their art-established usage levels. Thus, for example,the compositions may contain additional, compatible,pharmaceutically-active materials such as, for example, antipruritics,astringents, local anesthetics or anti-inflammatory agents, or maycontain additional materials useful in physically formulating variousdosage forms of the compositions of the present invention, such as dyes,flavoring agents, preservatives, antioxidants, opacifiers, thickeningagents and stabilizers. However, such materials, when added, should notunduly interfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

[0136] Aqueous suspensions may contain substances which increase theviscosity of the suspension including, for example, sodiumcarboxymethylcellulose, sorbitol and/or dextran. The suspension may alsocontain stabilizers.

[0137] Certain embodiments of the invention provide pharmaceuticalcompositions containing (a) one or more antisense compounds and (b) oneor more other chemotherapeutic agents which function by a non-antisensemechanism. Examples of such chemotherapeutic agents include but are notlimited to daunorubicin, daunomycin, dactinomycin, doxorubicin,epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide,cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C,actinomycin D, mithramycin, prednisone, hydroxyprogesterone,testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine,pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil,methylcyclohexylnitrosurea, nitrogen mustards, melphalan,cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine,5-azacytidine, hydroxyurea, deoxycoformycin,4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol,vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan,topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol(DES). See, generally, The Merck Manual of Diagnosis and Therapy, 15thEd. 1987, pp. 1206-1228, Berkow et al., eds., Rahway, N.J. When usedwith the compounds of the invention, such chemotherapeutic agents may beused individually (e.g., 5-FU and oligonucleotide), sequentially (e.g.,5-FU and oligonucleotide for a period of time followed by MTX andoligonucleotide), or in combination with one or more other suchchemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU,radiotherapy and oligonucleotide). Anti-inflammatory drugs, includingbut not limited to nonsteroidal anti-inflammatory drugs andcorticosteroids, and antiviral drugs, including but not limited toribivirin, vidarabine, acyclovir and ganciclovir, may also be combinedin compositions of the invention. See, generally, The Merck Manual ofDiagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway,N.J., pages 2499-2506 and 46-49, respectively). Other non-antisensechemotherapeutic agents are also within the scope of this invention. Twoor more combined compounds may be used together or sequentially.

[0138] In another related embodiment, compositions of the invention maycontain one or more antisense compounds, particularly oligonucleotides,targeted to a first nucleic acid and one or more additional antisensecompounds targeted to a second nucleic acid target. Numerous examples ofantisense compounds are known in the art. Two or more combined compoundsmay be used together or sequentially.

[0139] The formulation of therapeutic compositions and their subsequentadministration is believed to be within the skill of those in the art.Dosing is dependent on severity and responsiveness of the disease stateto be treated, with the course of treatment lasting from several days toseveral months, or until a cure is effected or a diminution of thedisease state is achieved. Optimal dosing schedules can be calculatedfrom measurements of drug accumulation in the body of the patient.Persons of ordinary skill can easily determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosages may vary dependingon the relative potency of individual oligonucleotides, and cangenerally be estimated based on EC₅₀s found to be effective in in vitroand in vivo animal models. In general, dosage is from 0.01 ug to 100 gper kg of body weight, and may be given once or more daily, weekly,monthly or yearly, or even once every 2 to 20 years. Persons of ordinaryskill in the art can easily estimate repetition rates for dosing basedon measured residence times and concentrations of the drug in bodilyfluids or tissues. Following successful treatment, it may be desirableto have the patient undergo maintenance therapy to prevent therecurrence of the disease state, wherein the oligonucleotide isadministered in maintenance doses, ranging from 0.01 ug to 100 g per kgof body weight, once or more daily, to once every 20 years.

[0140] While the present invention has been described with specificityin accordance with certain of its preferred embodiments, the followingexamples serve only to illustrate the invention and are not intended tolimit the same.

EXAMPLES Example 1 Nucleoside Phosphoramidites for OligonucleotideSynthesis Deoxy and 2′-alkoxy Amidites

[0141] 2′-Deoxy and 2′-methoxy beta-cyanoethyldiisopropylphosphoramidites were purchased from commercial sources (e.g. Chemgenes,Needham, Mass. or Glen Research, Inc. Sterling, Va.). Other 2′-O-alkoxysubstituted nucleoside amidites are prepared as described in U.S. Pat.No. 5,506,351, herein incorporated by reference. For oligonucleotidessynthesized using 2′-alkoxy amidites, optimized synthesis cycles weredeveloped that incorporate multiple steps coupling longer wait timesrelative to standard synthesis cycles.

[0142] The following abbreviations are used in the text: thin layerchromatography (TLC), melting point (MP), high pressure liquidchromatography (HPLC), Nuclear Magnetic Resonance (NMR), argon (Ar),methanol (MeOH), dichloromethane (CH₂Cl₂), triethylamine (TEA), dimethylformamide (DMF), ethyl acetate (EtOAc), dimethyl sulfoxide (DMSO),tetrahydrofuran (THF).

[0143] Oligonucleotides containing 5-methyl-2′-deoxycytidine (5-Me-dC)nucleotides were synthesized according to published methods (Sanghvi,et. al., Nucleic Acids Research, 1993, 21, 3197-3203) using commerciallyavailable phosphoramidites (Glen Research, Sterling, Va. or ChemGenes,Needham, Mass.) or prepared as follows:

[0144] Preparation of 5′-O-Dimethoxytrityl-thymidine Intermediate for5-methyl dC Amidite

[0145] To a 50 L glass reactor equipped with air stirrer and Ar gas linewas added thymidine (1.00 kg, 4.13 mol) in anhydrous pyridine (6 L) atambient temperature. Dimethoxytrityl (DMT) chloride (1.47 kg, 4.34 mol,1.05 eq) was added as a solid in four portions over 1 h. After 30 min,TLC indicated approx. 95% product, 2% thymidine, 5% DMT reagent andby-products and 2% 3′,5′-bis DMT product (R_(f) in EtOAc 0.45, 0.05,0.98, 0.95 respectively). Saturated sodium bicarbonate (4 L) and CH₂Cl₂were added with stirring (pH of the aqueous layer 7.5). An additional 18L of water was added, the mixture was stirred, the phases wereseparated, and the organic layer was transferred to a second 50 Lvessel. The aqueous layer was extracted with additional CH₂Cl₂ (2×2 L).The combined organic layer was washed with water (10 L) and thenconcentrated in a rotary evaporator to approx. 3.6 kg total weight. Thiswas redissolved in CH₂Cl₂ (3.5 L), added to the reactor followed bywater (6 L) and hexanes (13 L). The mixture was vigorously stirred andseeded to give a fine white suspended solid starting at the interface.After stirring for 1 h, the suspension was removed by suction through a½″ diameter teflon tube into a 20 L suction flask, poured onto a 25 cmCoors Buchner funnel, washed with water (2×3 L) and a mixture of hexanes—CH₂Cl₂ (4:1, 2×3 L) and allowed to air dry overnight in pans (1″ deep).This was further dried in a vacuum oven (75° C., 0.1 mm Hg, 48 h) to aconstant weight of 2072 g (93%) of a white solid, (mp 122-124° C.). TLCindicated a trace contamination of the bis DMT product. NMR spectroscopyalso indicated that 1-2 mole percent pyridine and about 5 mole percentof hexanes was still present.

[0146] Preparation of 5′-O-Dimethoxytrityl-2′-deoxy-5-methylcytidineIntermediate for 5-methyl-dC Amidite

[0147] To a 50 L Schott glass-lined steel reactor equipped with anelectric stirrer, reagent addition pump (connected to an additionfunnel), heating/cooling system, internal thermometer and an Ar gas linewas added 5′-O-dimethoxytrityl-thymidine (3.00 kg, 5.51 mol), anhydrousacetonitrile (25 L) and TEA (12.3 L, 88.4 mol, 16 eq). The mixture waschilled with stirring to −10° C. internal temperature (external −20°C.). Trimethylsilylchloride (2.1 L, 16.5 mol, 3.0 eq) was added over 30minutes while maintaining the internal temperature below −5° C.,followed by a wash of anhydrous acetonitrile (1 L). Note: the reactionis mildly exothermic and copious hydrochloric acid fumes form over thecourse of the addition. The reaction was allowed to warm to 0° C. andthe reaction progress was confirmed by TLC (EtOAc-hexanes 4:1; R_(f)0.43 to 0.84 of starting material and silyl product, respectively). Uponcompletion, triazole (3.05 kg, 44 mol, 8.0 eq) was added the reactionwas cooled to −20° C. internal temperature (external −30° C.).Phosphorous oxychloride (1035 mL, 11.1 mol, 2.01 eq) was added over 60min so as to maintain the temperature between −20° C. and −10° C. duringthe strongly exothermic process, followed by a wash of anhydrousacetonitrile (1 L). The reaction was warmed to 0° C. and stirred for 1h. TLC indicated a complete conversion to the triazole product (R_(f)0.83 to 0.34 with the product spot glowing in long wavelength UV light).The reaction mixture was a peach-colored thick suspension, which turneddarker red upon warming without apparent decomposition. The reaction wascooled to −15° C. internal temperature and water (5 L) was slowly addedat a rate to maintain the temperature below +10° C. in order to quenchthe reaction and to form a homogenous solution. (Caution: this reactionis initially very strongly exothermic). Approximately one-half of thereaction volume (22 L) was transferred by air pump to another vessel,diluted with EtOAc (12 L) and extracted with water (2×8 L). The combinedwater layers were back-extracted with EtOAc (6 L). The water layer wasdiscarded and the organic layers were concentrated in a 20 L rotaryevaporator to an oily foam. The foam was coevaporated with anhydrousacetonitrile (4 L) to remove EtOAc. (note: dioxane may be used insteadof anhydrous acetonitrile if dried to a hard foam). The second half ofthe reaction was treated in the same way. Each residue was dissolved indioxane (3 L) and concentrated ammonium hydroxide (750 mL) was added. Ahomogenous solution formed in a few minutes and the reaction was allowedto stand overnight (although the reaction is complete within 1 h).

[0148] TLC indicated a complete reaction (product R_(f) 0.35 inEtOAc-MeOH 4:1). The reaction solution was concentrated on a rotaryevaporator to a dense foam. Each foam was slowly redissolved in warmEtOAc (4 L; 50° C.), combined in a 50 L glass reactor vessel, andextracted with water (2×4L) to remove the triazole by-product. The waterwas back-extracted with EtOAc (2 L). The organic layers were combinedand concentrated to about 8 kg total weight, cooled to 0° C. and seededwith crystalline product. After 24 hours, the first crop was collectedon a 25 cm Coors Buchner funnel and washed repeatedly with EtOAc (3×3L)until a white powder was left and then washed with ethyl ether (2×3L).The solid was put in pans (1″ deep) and allowed to air dry overnight.The filtrate was concentrated to an oil, then redissolved in EtOAc (2L), cooled and seeded as before. The second crop was collected andwashed as before (with proportional solvents) and the filtrate was firstextracted with water (2×1L) and then concentrated to an oil. The residuewas dissolved in EtOAc (1 L) and yielded a third crop which was treatedas above except that more washing was required to remove a yellow oilylayer.

[0149] After air-drying, the three crops were dried in a vacuum oven(50° C., 0.1 mm Hg, 24 h) to a constant weight (1750, 600 and 200 g,respectively) and combined to afford 2550 g (85%) of a white crystallineproduct (MP 215-217° C.) when TLC and NMR spectroscopy indicated purity.The mother liquor still contained mostly product (as determined by TLC)and a small amount of triazole (as determined by NMR spectroscopy), bisDMT product and unidentified minor impurities. If desired, the motherliquor can be purified by silica gel chromatography using a gradient ofMeOH (0-25%) in EtOAc to further increase the yield.

[0150] Preparation of5′-O-Dimethoxytrityl-2′-deoxy-N4-benzoyl-5-methylcytidine PenultimateIntermediate for 5-methyl dC Amidite

[0151] Crystalline 5′-O-dimethoxytrityl-5-methyl-2′-deoxycytidine (2000g, 3.68 mol) was dissolved in anhydrous DMF (6.0 kg) at ambienttemperature in a 50 L glass reactor vessel equipped with an air stirrerand argon line. Benzoic anhydride (Chem Impex not Aldrich, 874 g, 3.86mol, 1.05 eq) was added and the reaction was stirred at ambienttemperature for 8 h. TLC (CH₂Cl₂-EtOAc; CH₂Cl₂-EtOAc 4:1; R_(f) 0.25)indicated approx. 92% complete reaction. An additional amount of benzoicanhydride (44 g, 0.19 mol) was added. After a total of 18 h, TLCindicated approx. 96% reaction completion. The solution was diluted withEtOAc (20 L), TEA (1020 mL, 7.36 mol, ca 2.0 eq) was added withstirring, and the mixture was extracted with water (15 L, then 2×10 L).The aqueous layer was removed (no back-extraction was needed) and theorganic layer was concentrated in 2×20 L rotary evaporator flasks untila foam began to form. The residues were coevaporated with acetonitrile(1.5 L each) and dried (0.1 mm Hg, 25° C., 24 h) to 2520 g of a densefoam. High pressure liquid chromatography (HPLC) revealed acontamination of 6.3% of N4, 3′-O-dibenzoyl product, but very littleother impurities.

[0152] The product was purified by Biotage column chromatography (5 kgBiotage) prepared with 65:35:1 hexanes-EtOAc-TEA (4L). The crude product(800 g),dissolved in CH₂Cl₂ (2 L), was applied to the column. The columnwas washed with the 65:35:1 solvent mixture (20 kg), then 20:80:1solvent mixture (10 kg), then 99:1 EtOAc:TEA (17 kg). The fractionscontaining the product were collected, and any fractions containing theproduct and impurities were retained to be resubjected to columnchromatography. The column was re-equilibrated with the original 65:35:1solvent mixture (17 kg). A second batch of crude product (840 g) wasapplied to the column as before. The column was washed with thefollowing solvent gradients: 65:35:1 (9 kg), 55:45:1 (20 kg), 20:80:1(10 kg), and 99:1 EtOAc:TEA(15 kg). The column was reequilibrated asabove, and a third batch of the crude product (850 g) plus impurefractions recycled from the two previous columns (28 g) was purifiedfollowing the procedure for the second batch. The fractions containingpure product combined and concentrated on a 20L rotary evaporator,co-evaporated with acetontirile (3 L) and dried (0.1 mm Hg, 48 h, 25°C.) to a constant weight of 2023 g (85%) of white foam and 20 g ofslightly contaminated product from the third run. HPLC indicated apurity of 99.8% with the balance as the diBenzoyl product.

[0153][5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N⁴-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(5-methyl dC amidite)

[0154]5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N⁴-benzoyl-5-methylcytidine(998 g, 1.5 mol) was dissolved in anhydrous DMF (2 L). The solution wasco-evaporated with toluene (300 ml) at 50° C. under reduced pressure,then cooled to room temperature and 2-cyanoethyltetraisopropylphosphorodiamidite (680 g, 2.26 mol) and tetrazole (52.5g, 0.75 mol) were added. The mixture was shaken until all tetrazole wasdissolved, N-methylimidazole (15 ml) was added and the mixture was leftat room temperature for 5 hours. TEA (300 ml) was added, the mixture wasdiluted with DMF (2.5 L) and water (600 ml), and extracted with hexane(3×3 L). The mixture was diluted with water (1.2 L) and extracted with amixture of toluene (7.5 L) and hexane (6 L). The two layers wereseparated, the upper layer was washed with DMF-water (7:3 v/v, 3×2 L)and water (3×2 L), and the phases were separated. The organic layer wasdried (Na₂SO₄), filtered and rotary evaporated. The residue wasco-evaporated with acetonitrile (2×2 L) under reduced pressure and driedto a constant weight (25° C., 0.1 mm Hg, 40 h) to afford 1250 g anoff-white foam solid (96%).

[0155] 2′-Fluoro Amidites

[0156] 2′-Fluorodeoxyadenosine Amidites

[0157] 2′-fluoro oligonucleotides were synthesized as describedpreviously [Kawasaki, et. al., J. Med. Chem., 1993, 36, 831-841] andU.S. Pat. No. 5,670,633, herein incorporated by reference. Thepreparation of 2′-fluoropyrimidines containing a 5-methyl substitutionare described in U.S. Pat. No. 5,861,493. Briefly, the protectednucleoside N6-benzoyl-2′-deoxy-2′-fluoroadenosine was synthesizedutilizing commercially available 9-beta-D-arabinofuranosyladenine asstarting material and whereby the 2′-alpha-fluoro atom is introduced bya S_(N)2-displacement of a 2′-beta-triflate group. ThusN6-benzoyl-9-beta-D-arabinofuranosyladenine was selectively protected inmoderate yield as the 3′,5′-ditetrahydropyranyl (THP) intermediate.Deprotection of the THP and N6-benzoyl groups was accomplished usingstandard methodologies to obtain the 5′-dimethoxytrityl-(DMT) and5′-DMT-3′-phosphoramidite intermediates.

[0158] 2′-Fluorodeoxyguanosine

[0159] The synthesis of 2′-deoxy-2′-fluoroguanosine was accomplishedusing tetraisopropyldisiloxanyl (TPDS) protected9-beta-D-arabinofuranosylguanine as starting material, and conversion tothe intermediate isobutyryl-arabinofuranosylguanosine. Alternatively,isobutyryl-arabinofuranosylguanosine was prepared as described by Rosset al., (Nucleosides & Nucleosides, 16, 1645, 1997). Deprotection of theTPDS group was followed by protection of the hydroxyl group with THP togive isobutyryl di-THP protected arabinofuranosylguanine. SelectiveO-deacylation and triflation was followed by treatment of the crudeproduct with fluoride, then deprotection of the THP groups. Standardmethodologies were used to obtain the 5′-DMT- and5′-DMT-3′-phosphoramidites.

[0160] 2′-Fluorouridine

[0161] Synthesis of 2′-deoxy-2′-fluorouridine was accomplished by themodification of a literature procedure in which2,2′-anhydro-1-beta-D-arabinofuranosyluracil was treated with 70%hydrogen fluoride-pyridine. Standard procedures were used to obtain the5′-DMT and 5′-DMT-3′phosphoramidites.

[0162] 2′-Fluorodeoxycytidine

[0163] 2′-deoxy-2′-fluorocytidine was synthesized via amination of2′-deoxy-2′-fluorouridine, followed by selective protection to giveN4-benzoyl-2′-deoxy-2′-fluorocytidine. Standard procedures were used toobtain the 5′-DMT and 5′-DMT-3′phosphoramidites.

[0164] 2′-O-(2-Methoxyethyl) Modified Amidites

[0165] 2′-O-Methoxyethyl-substituted nucleoside amidites (otherwiseknown as MOE amidites) are prepared as follows, or alternatively, as perthe methods of Martin, P., (Helvetica Chimica Acta, 1995, 78, 486-504).

[0166] Preparation of 2′-O-(2-methoxyethyl)-5-methyluridine Intermediate

[0167] 2,2′-Anhydro-5-methyl-uridine (2000 g, 8.32 mol),tris(2-methoxyethyl)borate (2504 g, 10.60 mol), sodium bicarbonate (60g, 0.70 mol) and anhydrous 2-methoxyethanol (5 L) were combined in a 12L three necked flask and heated to 130° C. (internal temp) atatmospheric pressure, under an argon atmosphere with stirring for 21 h.TLC indicated a complete reaction. The solvent was removed under reducedpressure until a sticky gum formed (50-85° C. bath temp and 100-11 mmHg) and the residue was redissolved in water (3 L) and heated to boilingfor 30 min in order the hydrolyze the borate esters. The water wasremoved under reduced pressure until a foam began to form and then theprocess was repeated. HPLC indicated about 77% product, 15% dimer (5′ ofproduct attached to 2′ of starting material) and unknown derivatives,and the balance was a single unresolved early eluting peak.

[0168] The gum was redissolved in brine (3 L), and the flask was rinsedwith additional brine (3 L). The combined aqueous solutions wereextracted with chloroform (20 L) in a heavier-than continuous extractorfor 70 h. The chloroform layer was concentrated by rotary evaporation ina 20 L flask to a sticky foam (2400 g). This was coevaporated with MeOH(400 mL) and EtOAc (8 L) at 75° C. and 0.65 atm until the foam dissolvedat which point the vacuum was lowered to about 0.5 atm. After 2.5 L ofdistillate was collected a precipitate began to form and the flask wasremoved from the rotary evaporator and stirred until the suspensionreached ambient temperature. EtOAc (2 L) was added and the slurry wasfiltered on a 25 cm table top Buchner funnel and the product was washedwith EtOAc (3×2 L). The bright white solid was air dried in pans for 24h then further dried in a vacuum oven (50° C., 0.1 mm Hg, 24 h) toafford 1649 g of a white crystalline solid (mp 115.5-116.5° C.).

[0169] The brine layer in the 20 L continuous extractor was furtherextracted for 72 h with recycled chloroform. The chloroform wasconcentrated to 120 g of oil and this was combined with the motherliquor from the above filtration (225 g), dissolved in brine (250 mL)and extracted once with chloroform (250 mL). The brine solution wascontinuously extracted and the product was crystallized as describedabove to afford an additional 178 g of crystalline product containingabout 2% of thymine. The combined yield was 1827 g (69.4%). HPLCindicated about 99.5% purity with the balance being the dimer.

[0170] Preparation of 5′-O-DMT-2′-O-(2-methoxyethyl)-5-methyluridinePenultimate Intermediate

[0171] In a 50 L glass-lined steel reactor,2′-O-(2-methoxyethyl)-5-methyl-uridine (MOE-T, 1500 g, 4.738 mol),lutidine (1015 g, 9.476 mol) were dissolved in anhydrous acetonitrile(15 L). The solution was stirred rapidly and chilled to −10° C.(internal temperature). Dimethoxytriphenylmethyl chloride (1765.7 g,5.21 mol) was added as a solid in one portion. The reaction was allowedto warm to −2° C. over 1 h. (Note: The reaction was monitored closely byTLC (EtOAc) to determine when to stop the reaction so as to not generatethe undesired bis-DMT substituted side product). The reaction wasallowed to warm from −2 to 3° C. over 25 min. then quenched by addingMeOH (300 mL) followed after 10 min by toluene (16 L) and water (16 L).The solution was transferred to a clear 50 L vessel with a bottomoutlet, vigorously stirred for 1 minute, and the layers separated. Theaqueous layer was removed and the organic layer was washed successivelywith 10% aqueous citric acid (8 L) and water (12 L). The product wasthen extracted into the aqueous phase by washing the toluene solutionwith aqueous sodium hydroxide (0.5N, 16 L and 8 L). The combined aqueouslayer was overlayed with toluene (12 L) and solid citric acid (8 moles,1270 g) was added with vigorous stirring to lower the pH of the aqueouslayer to 5.5 and extract the product into the toluene. The organic layerwas washed with water (10 L) and TLC of the organic layer indicated atrace of DMT-O-Me, bis DMT and dimer DMT.

[0172] The toluene solution was applied to a silica gel column (6 Lsintered glass funnel containing approx. 2 kg of silica gel slurriedwith toluene (2 L) and TEA(25 mL)) and the fractions were eluted withtoluene (12 L) and EtOAc (3×4 L) using vacuum applied to a filter flaskplaced below the column. The first EtOAc fraction containing both thedesired product and impurities were resubjected to column chromatographyas above. The clean fractions were combined, rotary evaporated to afoam, coevaporated with acetonitrile (6 L) and dried in a vacuum oven(0.1 mm Hg, 40 h, 40° C.) to afford 2850 g of a white crisp foam. NMRspectroscopy indicated a 0.25 mole % remainder of acetonitrile(calculates to be approx. 47 g) to give a true dry weight of 2803 g(96%). HPLC indicated that the product was 99.41% pure, with theremainder being 0.06 DMT-O-Me, 0.10 unknown, 0.44 bis DMT, and nodetectable dimer DMT or 3′-O-DMT.

[0173] Preparation of[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-5-methyluridin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE T Amidite)

[0174]5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-5-methyluridine(1237 g, 2.0 mol) was dissolved in anhydrous DMF (2.5 L). The solutionwas co-evaporated with toluene (200 ml) at 50° C. under reducedpressure, then cooled to room temperature and 2-cyanoethyltetraisopropylphosphorodiamidite (900 g, 3.0 mol) and tetrazole (70 g,1.0 mol) were added. The mixture was shaken until all tetrazole wasdissolved, N-methylimidazole (20 ml) was added and the solution was leftat room temperature for 5 hours. TEA (300 ml) was added, the mixture wasdiluted with DMF (3.5 L) and water (600 ml) and extracted with hexane(3×3L). The mixture was diluted with water (1.6 L) and extracted withthe mixture of toluene (12 L) and hexanes (9 L). The upper layer waswashed with DMF-water (7:3 v/v, 3×3 L) and water (3×3 L). The organiclayer was dried (Na₂SO₄), filtered and evaporated. The residue wasco-evaporated with acetonitrile (2×2 L) under reduced pressure and driedin a vacuum oven (25° C., 0.1 mm Hg, 40 h) to afford 1526 g of anoff-white foamy solid (95%).

[0175] Preparation of5′-O-Dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methylcytidine Intermediate

[0176] To a 50 L Schott glass-lined steel reactor equipped with anelectric stirrer, reagent addition pump (connected to an additionfunnel), heating/cooling system, internal thermometer and argon gas linewas added 5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methyl-uridine(2.616 kg, 4.23 mol, purified by base extraction only and no scrubcolumn), anhydrous acetonitrile (20 L), and TEA (9.5 L, 67.7 mol, 16eq). The mixture was chilled with stirring to −10° C. internaltemperature (external −20° C.). Trimethylsilylchloride (1.60 L, 12.7mol, 3.0 eq) was added over 30 min. while maintaining the internaltemperature below −5° C., followed by a wash of anhydrous acetonitrile(1 L). (Note: the reaction is mildly exothermic and copious hydrochloricacid fumes form over the course of the addition). The reaction wasallowed to warm to 0° C. and the reaction progress was confirmed by TLC(EtOAc, R_(f) 0.68 and 0.87 for starting material and silyl product,respectively). Upon completion, triazole (2.34 kg, 33.8 mol, 8.0 eq) wasadded the reaction was cooled to −20° C. internal temperature (external−30° C.). Phosphorous oxychloride (793 mL, 8.51 mol, 2.01 eq) was addedslowly over 60 min so as to maintain the temperature between −20° C. and−10° C. (note: strongly exothermic), followed by a wash of anhydrousacetonitrile (1 L). The reaction was warmed to 0° C. and stirred for 1h, at which point it was an off-white thick suspension. TLC indicated acomplete conversion to the triazole product (EtOAc, R_(f) 0.87 to 0.75with the product spot glowing in long wavelength UV light). The reactionwas cooled to −15° C. and water (5 L) was slowly added at a rate tomaintain the temperature below +10° C. in order to quench the reactionand to form a homogenous solution. (Caution: this reaction is initiallyvery strongly exothermic). Approximately one-half of the reaction volume(22 L) was transferred by air pump to another vessel, diluted with EtOAc(12 L) and extracted with water (2×8 L). The second half of the reactionwas treated in the same way. The combined aqueous layers wereback-extracted with EtOAc (8 L) The organic layers were combined andconcentrated in a 20 L rotary evaporator to an oily foam. The foam wascoevaporated with anhydrous acetonitrile (4 L) to remove EtOAc. (note:dioxane may be used instead of anhydrous acetonitrile if dried to a hardfoam). The residue was dissolved in dioxane (2 L) and concentratedammonium hydroxide (750 mL) was added. A homogenous solution formed in afew minutes and the reaction was allowed to stand overnight

[0177] TLC indicated a complete reaction (CH₂Cl₂-acetone-MeOH, 20:5:3,R_(f) 0.51). The reaction solution was concentrated on a rotaryevaporator to a dense foam and slowly redissolved in warm CH₂Cl₂ (4 L,40° C.) and transferred to a 20 L glass extraction vessel equipped witha air-powered stirrer. The organic layer was extracted with water (2×6L) to remove the triazole by-product. (Note: In the first extraction anemulsion formed which took about 2 h to resolve). The water layer wasback-extracted with CH₂Cl₂ (2×2 L), which in turn was washed with water(3 L). The combined organic layer was concentrated in 2×20 L flasks to agum and then recrystallized from EtOAc seeded with crystalline product.After sitting overnight, the first crop was collected on a 25 cm CoorsBuchner funnel and washed repeatedly with EtOAc until a whitefree-flowing powder was left (about 3×3 L). The filtrate wasconcentrated to an oil recrystallized from EtOAc, and collected asabove. The solid was air-dried in pans for 48 h, then further dried in avacuum oven (50° C., 0.1 mm Hg, 17 h) to afford 2248 g of a brightwhite, dense solid (86%). An HPLC analysis indicated both crops to be99.4% pure and NMR spectroscopy indicated only a faint trace of EtOAcremained.

[0178] Preparation of5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-N4-benzoyl-5-methyl-cytidinePenultimate Intermediate:

[0179] Crystalline5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methyl-cytidine (1000 g,1.62 mol) was suspended in anhydrous DMF (3 kg) at ambient temperatureand stirred under an Ar atmosphere. Benzoic anhydride (439.3 g, 1.94mol) was added in one portion. The solution clarified after 5 hours andwas stirred for 16 h. HPLC indicated 0.45% starting material remained(as well as 0.32% N4, 3′-O-bis Benzoyl). An additional amount of benzoicanhydride (6.0 g, 0.0265 mol) was added and after 17 h, HPLC indicatedno starting material was present. TEA (450 mL, 3.24 mol) and toluene (6L) were added with stirring for 1 minute. The solution was washed withwater (4×4 L), and brine (2×4 L). The organic layer was partiallyevaporated on a 20 L rotary evaporator to remove 4 L of toluene andtraces of water. HPLC indicated that the bis benzoyl side product waspresent as a 6% impurity. The residue was diluted with toluene (7 L) andanhydrous DMSO (200 mL, 2.82 mol) and sodium hydride (60% in oil, 70 g,1.75 mol) was added in one portion with stirring at ambient temperatureover 1 h. The reaction was quenched by slowly adding then washing withaqueous citric acid (10%, 100 mL over 10 min, then 2×4 L), followed byaqueous sodium bicarbonate (2%, 2 L), water (2×4 L) and brine (4 L). Theorganic layer was concentrated on a 20 L rotary evaporator to about 2 Ltotal volume. The residue was purified by silica gel columnchromatography (6 L Buchner funnel containing 1.5 kg of silica gelwetted with a solution of EtOAc-hexanes-TEA (70:29:1)). The product waseluted with the same solvent (30 L) followed by straight EtOAc (6 L).The fractions containing the product were combined, concentrated on arotary evaporator to a foam and then dried in a vacuum oven (50° C., 0.2mm Hg, 8 h) to afford 1155 g of a crisp, white foam (98%). HPLCindicated a purity of >99.7%.

[0180] Preparation of[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE 5-Me-C Amidite)

[0181]5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methylcytidine(1082 g, 1.5 mol) was dissolved in anhydrous DMF (2 L) and co-evaporatedwith toluene (300 ml) at 50° C. under reduced pressure. The mixture wascooled to room temperature and 2-cyanoethyltetraisopropylphosphorodiamidite (680 g, 2.26 mol) and tetrazole (52.5g, 0.75 mol) were added. The mixture was shaken until all tetrazole wasdissolved, N-methylimidazole (30 ml) was added, and the mixture was leftat room temperature for 5 hours. TEA (300 ml) was added, the mixture wasdiluted with DMF (1 L) and water (400 ml) and extracted with hexane (3×3L). The mixture was diluted with water (1.2 L) and extracted with amixture of toluene (9 L) and hexanes (6 L). The two layers wereseparated and the upper layer was washed with DMF-water (60:40 v/v, 3×3L) and water (3×2 L). The organic layer was dried (Na₂SO₄), filtered andevaporated. The residue was co-evaporated with acetonitrile (2×2 L)under reduced pressure and dried in a vacuum oven (25° C., 0.1 mm Hg, 40h) to afford 1336 g of an off-white foam (97%).

[0182] Preparation of[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁶-benzoyladenosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE A Amdite)

[0183]5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁶-benzoyladenosine(purchased from Reliable Biopharmaceutical, St. Lois, Mo.), 1098 g, 1.5mol) was dissolved in anhydrous DMF (3 L) and co-evaporated with toluene(300 ml) at 50° C. The mixture was cooled to room temperature and2-cyanoethyl tetraisopropylphosphorodiamidite (680 g, 2.26 mol) andtetrazole (78.8 g, 1.24 mol) were added. The mixture was shaken untilall tetrazole was dissolved, N-methylimidazole (30 ml) was added, andmixture was left at room temperature for 5 hours. TEA (300 ml) wasadded, the mixture was diluted with DMF (1 L) and water (400 ml) andextracted with hexanes (3×3 L). The mixture was diluted with water (1.4L) and extracted with the mixture of toluene (9 L) and hexanes (6 L).The two layers were separated and the upper layer was washed withDMF-water (60:40, v/v, 3×3 L) and water (3×2 L). The organic layer wasdried (Na₂SO₄), filtered and evaporated to a sticky foam. The residuewas co-evaporated with acetonitrile (2.5 L) under reduced pressure anddried in a vacuum oven (25° C., 0.1 mm Hg, 40 h) to afford 1350 g of anoff-white foam solid (96%).

[0184] Prepartion of[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-isobutyrylguanosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE G Amidite)

[0185]5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-isobutyrlguanosine(purchased from Reliable Biopharmaceutical, St. Louis, Mo., 1426 g, 2.0mol) was dissolved in anhydrous DMF (2 L). The solution wasco-evaporated with toluene (200 ml) at 50° C., cooled to roomtemperature and 2-cyanoethyl tetraisopropylphosphorodiamidite (900 g,3.0 mol) and tetrazole (68 g, 0.97 mol) were added. The mixture wasshaken until all tetrazole was dissolved, N-methylimidazole (30 ml) wasadded, and the mixture was left at room temperature for 5 hours. TEA(300 ml) was added, the mixture was diluted with DMF (2 L) and water(600 ml) and extracted with hexanes (3×3 L). The mixture was dilutedwith water (2 L) and extracted with a mixture of toluene (10 L) andhexanes (5 L). The two layers were separated and the upper layer waswashed with DMF-water (60:40, v/v, 3×3 L). EtOAc (4 L) was added and thesolution was washed with water (3×4 L). The organic layer was dried(Na₂SO₄), filtered and evaporated to approx. 4 kg. Hexane (4 L) wasadded, the mixture was shaken for 10 min, and the supernatant liquid wasdecanted. The residue was co-evaporated with acetonitrile (2×2 L) underreduced pressure and dried in a vacuum oven (25° C., 0.1 mm Hg, 40 h) toafford 1660 g of an off-white foamy solid (91%).

[0186] 2′-O-(Aminooxyethyl) Nucleoside Amidites and2′-O-(dimethylaminooxyethyl) Nucleoside Amidites

[0187] 2′-(Dimethylaminooxyethoxy) Nucleoside Amidites

[0188] 2′-(Dimethylaminooxyethoxy) nucleoside amidites (also known inthe art as 2′-O-(dimethylaminooxyethyl) nucleoside amidites) areprepared as described in the following paragraphs. Adenosine, cytidineand guanosine nucleoside amidites are prepared similarly to thethymidine (5-methyluridine) except the exocyclic amines are protectedwith a benzoyl moiety in the case of adenosine and cytidine and withisobutyryl in the case of guanosine.

[0189] 5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine

[0190] O²-2′-anhydro-5-methyluridine (Pro. Bio. Sint., Varese, Italy,100.0 g, 0.416 mmol), dimethylaminopyridine (0.66 g, 0.013 eq, 0.0054mmol) were dissolved in dry pyridine (500 ml) at ambient temperatureunder an argon atmosphere and with mechanical stirring.tert-Butyldiphenylchlorosilane (125.8 g, 119.0 mL, 1.1 eq, 0.458 mmol)was added in one portion. The reaction was stirred for 16 h at ambienttemperature. TLC (R_(f) 0.22, EtOAc) indicated a complete reaction. Thesolution was concentrated under reduced pressure to a thick oil. Thiswas partitioned between CH₂Cl₂ (1 L) and saturated sodium bicarbonate(2×1 L) and brine (1 L). The organic layer was dried over sodiumsulfate, filtered, and concentrated under reduced pressure to a thickoil. The oil was dissolved in a 1:1 mixture of EtOAc and ethyl ether(600 mL) and cooling the solution to −10° C. afforded a whitecrystalline solid which was collected by filtration, washed with ethylether (3×200 mL) and dried (40° C., 1 mm Hg, 24 h) to afford 149 g ofwhite solid (74.8%). TLC and NMR spectroscopy were consistent with pureproduct.

[0191] 5′-O-tert-Butyldiphenylsilyl-2-O-(2-hydroxyethyl)-5-methyluridine

[0192] In the fume hood, ethylene glycol (350 mL, excess) was addedcautiously with manual stirring to a 2 L stainless steel pressurereactor containing borane in tetrahydrofuran (1.0 M, 2.0 eq, 622 mL).(Caution: evolves hydrogen gas).5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine (149 g, 0.311mol) and sodium bicarbonate (0.074 g, 0.003 eq) were added with manualstirring. The reactor was sealed and heated in an oil bath until aninternal temperature of 160° C. was reached and then maintained for 16 h(pressure <100 psig). The reaction vessel was cooled to ambienttemperature and opened. TLC (EtOAc, R_(f) 0.67 for desired product andR_(f) 0.82 for ara-T side product) indicated about 70% conversion to theproduct. The solution was concentrated under reduced pressure (10 to 1mm Hg) in a warm water bath (40-100° C.) with the more extremeconditions used to remove the ethylene glycol. (Alternatively, once theTHF has evaporated the solution can be diluted with water and theproduct extracted into EtOAc). The residue was purified by columnchromatography (2 kg silica gel, EtOAc-hexanes gradient 1:1 to 4:1). Theappropriate fractions were combined, evaporated and dried to afford 84 gof a white crisp foam (50%), contaminated starting material (17.4 g, 12%recovery) and pure reusable starting material (20 g, 13% recovery). TLCand NMR spectroscopy were consistent with 99% pure product.

[0193]2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine

[0194]5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine (20g, 36.98 mmol) was mixed with triphenylphosphine (11.63 g, 44.36 mmol)and N-hydroxyphthalimide (7.24 g, 44.36 mmol) and dried over P₂O₅ underhigh vacuum for two days at 40° C. The reaction mixture was flushed withargon and dissolved in dry THF (369.8 mL, Aldrich, sure seal bottle).Diethyl-azodicarboxylate (6.98 mL, 44.36 mmol) was added dropwise to thereaction mixture with the rate of addition maintained such that theresulting deep red coloration is just discharged before adding the nextdrop. The reaction mixture was stirred for 4 hrs., after which time TLC(EtOAc:hexane, 60:40) indicated that the reaction was complete. Thesolvent was evaporated in vacuuo and the residue purified by flashcolumn chromatography (eluted with 60:40 EtOAc:hexane), to yield2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine aswhite foam (21.819 g, 86%) upon rotary evaporation.

[0195]5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine

[0196]2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine(3.1 g, 4.5 mmol) was dissolved in dry CH₂Cl₂ (4.5 mL) andmethylhydrazine (300 mL, 4.64 mmol) was added dropwise at −10° C. to 0°C. After 1 h the mixture was filtered, the filtrate washed with ice coldCH₂Cl₂, and the combined organic phase was washed with water and brineand dried (anhydrous Na₂SO₄). The solution was filtered and evaporatedto afford 2′-O-(aminooxyethyl) thymidine, which was then dissolved inMeOH (67.5 mL). Formaldehyde (20% aqueous solution, w/w, 1.1 eq.) wasadded and the resulting mixture was stirred for 1 h. The solvent wasremoved under vacuum and the residue was purified by columnchromatography to yield5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine as white foam (1.95 g, 78%) upon rotaryevaporation.

[0197] 5′-O-tert-Butyldiphenylsilyl-2′-O-[N,Ndimethylaminooxyethyl]-5-methyluridine

[0198]5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine(1.77 g, 3.12 mmol) was dissolved in a solution of 1M pyridiniump-toluenesulfonate (PPTS) in dry MeOH (30.6 mL) and cooled to 10° C.under inert atmosphere. Sodium cyanoborohydride (0.39 g, 6.13 mmol) wasadded and the reaction mixture was stirred. After 10 minutes thereaction was warmed to room temperature and stirred for 2 h. while theprogress of the reaction was monitored by TLC (5% MeOH in CH₂Cl₂).Aqueous NaHCO₃ solution (5%, 10 mL) was added and the product wasextracted with EtOAc (2×20 mL). The organic phase was dried overanhydrous Na₂SO₄, filtered, and evaporated to dryness. This entireprocedure was repeated with the resulting residue, with the exceptionthat formaldehyde (20% w/w, 30 mL, 3.37 mol) was added upon dissolutionof the residue in the PPTS/MeOH solution. After the extraction andevaporation, the residue was purified by flash column chromatography and(eluted with 5% MeOH in CH₂Cl₂) to afford5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridineas a white foam (14.6 g, 80%) upon rotary evaporation.

[0199] 2′-O-(dimethylaminooxyethyl)-5-methyluridine

[0200] Triethylamine trihydrofluoride (3.91 mL, 24.0 mmol) was dissolvedin dry THF and TEA (1.67 mL, 12 mmol, dry, stored over KOH) and added to5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine(1.40 g, 2.4mmol). The reaction was stirred at room temperature for 24hrs and monitored by TLC (5% MeOH in CH₂Cl₂). The solvent was removedunder vacuum and the residue purified by flash column chromatography(eluted with 10% MeOH in CH₂Cl₂) to afford2′-O-(dimethylaminooxyethyl)-5-methyluridine (766 mg, 92.5%) upon rotaryevaporation of the solvent.

[0201] 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine

[0202] 2′-O-(dimethylaminooxyethyl)-5-methyluridine (750 mg, 2.17 mmol)was dried over P₂O₅ under high vacuum overnight at 40° C., co-evaporatedwith anhydrous pyridine (20 mL), and dissolved in pyridine (11 mL) underargon atmosphere. 4-dimethylaminopyridine (26.5 mg, 2.60 mmol) and4,4′-dimethoxytrityl chloride (880 mg, 2.60 mmol) were added to thepyridine solution and the reaction mixture was stirred at roomtemperature until all of the starting material had reacted. Pyridine wasremoved under vacuum and the residue was purified by columnchromatography (eluted with 10% MeOH in CH₂Cl₂ containing a few drops ofpyridine) to yield5′-O-DMT-2′-O-(dimethylamino-oxyethyl)-5-methyluridine (1.13 g, 80%)upon rotary evaporation.

[0203]5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

[0204] 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine (1.08 g,1.67 mmol) was co-evaporated with toluene (20 mL), N,N-diisopropylaminetetrazonide (0.29 g, 1.67 mmol) was added and the mixture was dried overP₂O₅ under high vacuum overnight at 40° C. This was dissolved inanhydrous acetonitrile (8.4 mL) and2-cyanoethyl-N,N,N¹,N¹-tetraisopropylphosphoramidite (2.12 mL, 6.08mmol) was added. The reaction mixture was stirred at ambient temperaturefor 4 h under inert atmosphere. The progress of the reaction wasmonitored by TLC (hexane:EtOAc 1:1). The solvent was evaporated, thenthe residue was dissolved in EtOAc (70 mL) and washed with 5% aqueousNaHCO₃ (40 mL) The EtOAc layer was dried over anhydrous Na₂SO₄,filtered, and concentrated. The residue obtained was purified by columnchromatography (EtOAc as eluent) to afford5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]asa foam (1.04 g, 74.9%) upon rotary evaporation.

[0205] 2′-(Aminooxyethoxy) Nucleoside Amidites

[0206] 2′-(Aminooxyethoxy) nucleoside amidites (also known in the art as2′-O-(aminooxyethyl) nucleoside amidites) are prepared as described inthe following paragraphs. Adenosine, cytidine and thymidine nucleosideamidites are prepared similarly.

[0207]N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

[0208] The 2′-O-aminooxyethyl guanosine analog may be obtained byselective 2′-O-alkylation of diaminopurine riboside. Multigramquantities of diaminopurine riboside may be purchased from Schering AG(Berlin) to provide 2′-O-(2-ethylacetyl) diaminopurine riboside alongwith a minor amount of the 3′-O-isomer. 2′-O-(2-ethylacetyl)diaminopurine riboside may be resolved and converted to2′-O-(2-ethylacetyl) guanosine by treatment with adenosine deaminase.(McGee, D. P. C., Cook, P. D., Guinosso, C. J., WO 94/02501 A1 940203.)Standard protection procedures should afford2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine and2-N-isobutyryl-6-O-diphenylcarbamoyl-2-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosinewhich may be reduced to provide2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-hydroxyethyl)-5′-O-(4,4′-dimethoxytrityl)guanosine.As before the hydroxyl group may be displaced by N-hydroxyphthalimidevia a Mitsunobu reaction, and the protected nucleoside may bephosphitylated as usual to yield2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-([2-phthalmidoxy]ethyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite].

[0209] 2′-dimethylaminoethoxyethoxy (2′-DMAEOE) Nucleoside Amidites

[0210] 2′-dimethylaminoethoxyethoxy nucleoside amidites (also known inthe art as 2′-O-dimethylaminoethoxyethyl, i.e., 2′—O—CH₂—O—CH₂—N(CH₂)₂,or 2′-DMAEOE nucleoside amidites) are prepared as follows. Othernucleoside amidites are prepared similarly.

[0211] 2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl Uridine

[0212] 2[2-(Dimethylamino)ethoxy]ethanol (Aldrich, 6.66 g, 50 mmol) wasslowly added to a solution of borane in tetra-hydrofuran (1 M, 10 mL, 10mmol) with stirring in a 100 mL bomb. (Caution: Hydrogen gas evolves asthe solid dissolves). O²-,2′-anhydro-5-methyluridine (1.2 g, 5 mmol),and sodium bicarbonate (2.5 mg) were added and the bomb was sealed,placed in an oil bath and heated to 155° C. for 26 h. then cooled toroom temperature. The crude solution was concentrated, the residue wasdiluted with water (200 mL) and extracted with hexanes (200 mL). Theproduct was extracted from the aqueous layer with EtOAc (3×200 mL) andthe combined organic layers were washed once with water, dried overanhydrous sodium sulfate, filtered and concentrated. The residue waspurified by silica gel column chromatography (eluted with 5:100:2MeOH/CH₂Cl₂/TEA) as the eluent. The appropriate fractions were combinedand evaporated to afford the product as a white solid.

[0213] 5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyl Uridine

[0214] To 0.5 g (1.3 mmol) of2′-O-[2(2-N,N-dimethylamino-ethoxy)ethyl)]-5-methyl uridine in anhydrouspyridine (8 mL), was added TEA (0.36 mL) and dimethoxytrityl chloride(DMT-Cl, 0.87 g, 2 eq.) and the reaction was stirred for 1 h. Thereaction mixture was poured into water (200 mL) and extracted withCH₂Cl₂ (2×200 mL). The combined CH₂Cl₂ layers were washed with saturatedNaHCO₃ solution, followed by saturated NaCl solution, dried overanhydrous sodium sulfate, filtered and evaporated. The residue waspurified by silica gel column chromatography (eluted with 5:100:1MeOH/CH₂Cl₂/TEA) to afford the product.

[0215]5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methylUridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite

[0216] Diisopropylaminotetrazolide (0.6 g) and2-cyanoethoxy-N,N-diisopropyl phosphoramidite (1.1 mL, 2 eq.) were addedto a solution of5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyluridine(2.17 g, 3 mmol) dissolved in CH₂Cl₂ (20 mL) under an atmosphere ofargon. The reaction mixture was stirred overnight and the solventevaporated. The resulting residue was purified by silica gel columnchromatography with EtOAc as the eluent to afford the title compound.

Example 2 Oligonucleotide Synthesis

[0217] Unsubstituted and substituted phosphodiester (P═O)oligonucleotides are synthesized on an automated DNA synthesizer(Applied Biosystems model 394) using standard phosphoramidite chemistrywith oxidation by iodine.

[0218] Phosphorothioates (P═S) are synthesized similar to phosphodiesteroligonucleotides with the following exceptions: thiation was effected byutilizing a 10% w/v solution of 3H-1,2-benzodithiole-3-one 1,1-dioxidein acetonitrile for the oxidation of the phosphite linkages. Thethiation reaction step time was increased to 180 sec and preceded by thenormal capping step. After cleavage from the CPG column and deblockingin concentrated ammonium hydroxide at 55° C. (12-16 hr), theoligonucleotides were recovered by precipitating with >3 volumes ofethanol from a 1 M NH₄OAc solution. Phosphinate oligonucleotides areprepared as described in U.S. Pat. No. 5,508,270, herein incorporated byreference.

[0219] Alkyl phosphonate oligonucleotides are prepared as described inU.S. Pat. No. 4,469,863, herein incorporated by reference.

[0220] 3′-Deoxy-3′-methylene phosphonate oligonucleotides are preparedas described in U.S. Pat. Nos. 5,610,289 or 5,625,050, hereinincorporated by reference.

[0221] Phosphoramidite oligonucleotides are prepared as described inU.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporatedby reference.

[0222] Alkylphosphonothioate oligonucleotides are prepared as describedin published PCT applications PCT/US94/00902 and PCT/US93/06976(published as WO 94/17093 and WO 94/02499, respectively), hereinincorporated by reference.

[0223] 3′-Deoxy-3′-amino phosphoramidate oligonucleotides are preparedas described in U.S. Pat. No. 5,476,925, herein incorporated byreference.

[0224] Phosphotriester oligonucleotides are prepared as described inU.S. Pat. No. 5,023,243, herein incorporated by reference.

[0225] Borano phosphate oligonucleotides are prepared as described inU.S. Pat. Nos. 5,130,302 and 5,177,198, both herein incorporated byreference.

Example 3 Oligonucleoside Synthesis

[0226] Methylenemethylimino linked oligonucleosides, also identified asMMI linked oligonucleosides, methylenedimethyl-hydrazo linkedoligonucleosides, also identified as MDH linked oligonucleosides, andmethylenecarbonylamino linked oligonucleosides, also identified asamide-3 linked oligonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified as amide-4 linked oligonucleosides, aswell as mixed backbone compounds having, for instance, alternating MMIand P═O or P═S linkages are prepared as described in U.S. Pat. Nos.5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of whichare herein incorporated by reference.

[0227] Formacetal and thioformacetal linked oligonucleosides areprepared as described in U.S. Pat. Nos.5,264,562 and 5,264,564, hereinincorporated by reference.

[0228] Ethylene oxide linked oligonucleosides are prepared as describedin U.S. Pat. No. 5,223,618, herein incorporated by reference.

Example 4 PNA Synthesis

[0229] Peptide nucleic acids (PNAs) are prepared in accordance with anyof the various procedures referred to in Peptide Nucleic Acids (PNA):Synthesis, Properties and Potential Applications, Bioorganic & MedicinalChemistry, 1996, 4, 5-23. They may also be prepared in accordance withU.S. Pat. Nos. 5,539,082, 5,700,922, and 5,719,262, herein incorporatedby reference.

Example 5 Synthesis of Chimeric Oligonucleotides

[0230] Chimeric oligonucleotides, oligonucleosides or mixedoligonucleotides/oligonucleosides of the invention can be of severaldifferent types. These include a first type wherein the “gap” segment oflinked nucleosides is positioned between 5′ and 3′ “wing” segments oflinked nucleosides and a second “open end” type wherein the “gap”segment is located at either the 3′ or the 5′ terminus of the oligomericcompound. Oligonucleotides of the first type are also known in the artas “gapmers” or gapped oligonucleotides. Oligonucleotides of the secondtype are also known in the art as “hemimers” or “wingmers”.

[0231] [2′-O-Me]--[2′-deoxy]--[2′-O-Me] Chimeric PhosphorothioateOligonucleotides

[0232] Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and2′-deoxy phosphorothioate oligonucleotide segments are synthesized usingan Applied Biosystems automated DNA synthesizer Model 394, as above.Oligonucleotides are synthesized using the automated synthesizer and2′-deoxy-5′-dimethoxytrityl-3′-O-phosphoramidite for the DNA portion and5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′and 3′wings.The standard synthesis cycle is modified by incorporating coupling stepswith increased reaction times for the5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite. The fully protectedoligonucleotide is cleaved from the support and deprotected inconcentrated ammonia (NH₄OH) for 12-16 hr at 55° C. The deprotectedoligo is then recovered by an appropriate method (precipitation, columnchromatography, volume reduced in vacuo and analyzedspetrophotometrically for yield and for purity by capillaryelectrophoresis and by mass spectrometry.

[0233] [2′-O-(2-Methoxyethyl)]--[2′-deoxy]--[2′-O-(Methoxyethyl)]Chimeric Phosphorothioate Oligonucleotides

[0234] [2′-O-(2-methoxyethyl)]--[2′-deoxy]--[-2′-O-(methoxyethyl)]chimeric phosphorothioate oligonucleotides were prepared as per theprocedure above for the 2′-O-methyl chimeric oligonucleotide, with thesubstitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methylamidites.

[0235] [2′-O-(2-Methoxyethyl)Phosphodiester]--[2′-deoxyPhosphorothioate]--[2′-O-(2-Methoxyethyl) Phosphodiester] ChimericOligonucleotides

[0236] [2′-O-(2-methoxyethyl phosphodiester]--[2′-deoxyphosphorothioate]--[2′-O-(methoxyethyl) phosphodiester] chimericoligonucleotides are prepared as per the above procedure for the2′-O-methyl chimeric oligonucleotide with the substitution of2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites, oxidationwith iodine to generate the phosphodiester internucleotide linkageswithin the wing portions of the chimeric structures and sulfurizationutilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) togenerate the phosphorothioate internucleotide linkages for the centergap.

[0237] Other chimeric oligonucleotides, chimeric oligonucleosides andmixed chimeric oligonucleotides/oligonucleosides are synthesizedaccording to U.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 6 Oligonucleotide Isolation

[0238] After cleavage from the controlled pore glass solid support anddeblocking in concentrated ammonium hydroxide at 55° C. for 12-16 hours,the oligonucleotides or oligonucleosides are recovered by precipitationout of 1 M NH₄OAc with >3 volumes of ethanol. Synthesizedoligonucleotides were analyzed by electrospray mass spectroscopy(molecular weight determination) and by capillary gel electrophoresisand judged to be at least 70% full length material. The relative amountsof phosphorothioate and phosphodiester linkages obtained in thesynthesis was determined by the ratio of correct molecular weightrelative to the −16 amu product (+/−32+/−48). For some studiesoligonucleotides were purified by HPLC, as described by Chiang et al.,J. Biol. Chem. 1991, 266, 18162-18171. Results obtained withHPLC-purified material were similar to those obtained with non-HPLCpurified material.

Example 7 Oligonucleotide Synthesis—96 Well Plate Format

[0239] Oligonucleotides were synthesized via solid phase P(III)phosphoramidite chemistry on an automated synthesizer capable ofassembling 96 sequences simultaneously in a 96-well format.Phosphodiester internucleotide linkages were afforded by oxidation withaqueous iodine. Phosphorothioate internucleotide linkages were generatedby sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide(Beaucage Reagent) in anhydrous acetonitrile. Standard base-protectedbeta-cyanoethyl-diiso-propyl phosphoramidites were purchased fromcommercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., orPharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesizedas per standard or patented methods. They are utilized as base protectedbeta-cyanoethyldiisopropyl phosphoramidites.

[0240] Oligonucleotides were cleaved from support and deprotected withconcentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hoursand the released product then dried in vacuo. The dried product was thenre-suspended in sterile water to afford a master plate from which allanalytical and test plate samples are then diluted utilizing roboticpipettors.

Example 8 Oligonucleotide Analysis—96-Well Plate Format

[0241] The concentration of oligonucleotide in each well was assessed bydilution of samples and UV absorption spectroscopy. The full-lengthintegrity of the individual products was evaluated by capillaryelectrophoresis (CE) in either the 96-well format (Beckman P/ACE™ MDQ)or, for individually prepared samples, on a commercial CE apparatus(e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition wasconfirmed by mass analysis of the compounds utilizing electrospray-massspectroscopy. All assay test plates were diluted from the master plateusing single and multi-channel robotic pipettors. Plates were judged tobe acceptable if at least 85% of the compounds on the plate were atleast 85% full length.

Example 9 Cell Culture and Oligonucleotide Treatment

[0242] The effect of antisense compounds on target nucleic acidexpression can be tested in any of a variety of cell types provided thatthe target nucleic acid is present at measurable levels. This can beroutinely determined using, for example, PCR or Northern blot analysis.The following cell types are provided for illustrative purposes, butother cell types can be routinely used, provided that the target isexpressed in the cell type chosen. This can be readily determined bymethods routine in the art, for example Northern blot analysis,ribonuclease protection assays, or RT-PCR.

[0243] T-24 Cells:

[0244] The human transitional cell bladder carcinoma cell line T-24 wasobtained from the American Type Culture Collection (ATCC) (Manassas,Va.). T-24 cells were routinely cultured in complete McCoy's 5A basalmedia (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10%fetal calf serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin100 units per mL, and streptomycin 100 micrograms per mL (InvitrogenCorporation, Carlsbad, Calif.). Cells were routinely passaged bytrypsinization and dilution when they reached 90% confluence. Cells wereseeded into 96-well plates (Falcon-Primaria #3872) at a density of 7000cells/well for use in RT-PCR analysis.

[0245] For Northern blotting or other analysis, cells may be seeded onto100 mm or other standard tissue culture plates and treated similarly,using appropriate volumes of medium and oligonucleotide.

[0246] A549 Cells:

[0247] The human lung carcinoma cell line A549 was obtained from theAmerican Type Culture Collection (ATCC) (Manassas, Va.). A549 cells wereroutinely cultured in DMEM basal media (Invitrogen Corporation,Carlsbad, Calif.) supplemented with 10% fetal calf serum (InvitrogenCorporation, Carlsbad, Calif.), penicillin 100 units per mL, andstreptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad,Calif.). Cells were routinely passaged by trypsinization and dilutionwhen they reached 90% confluence.

[0248] NHDF Cells:

[0249] Human neonatal dermal fibroblast (NHDF) were obtained from theClonetics Corporation (Walkersville, Md.). NHDFs were routinelymaintained in Fibroblast Growth Medium (Clonetics Corporation,Walkersville, Md.) supplemented as recommended by the supplier. Cellswere maintained for up to 10 passages as recommended by the supplier.

[0250] HEK Cells:

[0251] Human embryonic keratinocytes (HEK) were obtained from theClonetics Corporation (Walkersville, Md.). HEKs were routinelymaintained in Keratinocyte Growth Medium (Clonetics Corporation,Walkersville, Md.) formulated as recommended by the supplier. Cells wereroutinely maintained for up to 10 passages as recommended by thesupplier.

[0252] Jurkat Cells:

[0253] The human Jurkat cell line was obtained from the American TypeCulture Collection (ATCC) (Manassas, Va.). Jurkat cells were routinelycultured in RPMI Medium 1640 (Gibco/Life Technologies, Gaithersburg,Md.) supplemented with 20% fetal calf serum (Gibco/Life Technologies,Gaithersburg, Md.) Cells were routinely passaged by aspirating mediathat contained excess cells and replenishing with new media.

[0254] For electroporation, cells were diluted to 5×10⁶ cells/mL andplaced into 1 mm electroporation cuvettes. Electroporation is performedby treating with 10 μM oligonucleotide, at 90 Volts for 9 msec. Theentire electroporated samples are then placed into 500 μL of RPMI Medium1640 in 24-well plates. Plates are then left overnight.

[0255] Each sample was then divided into two 250 μL samples and spundown on poly-D-lysine (Sigma) coated 96-well plates (Falcon). Cells werethen washed and lysed in 150 μL RLT. Following the lysis step, RNA fromthe cells was isolated and quantitated as described in other examplesherein.

[0256] Differentiated 3T3-L1 Cells:

[0257] The mouse embryonic adipocyte cell line 3T3-L1 was obtained fromthe American Type Culture Collection (Manassas, Va.). 3T3-L1 cells weredifferentiated by culturing for three days in the presence of 400 nMinsulin, 250 nM dexamethasone and 0.5 mM IBMX (Sigma). Differentiated3T3L1 cells were then routinely cultured in DMEM, high glucose(Gibco/Life Technologies, Gaithersburg, Md.) supplemented with 10% fetalcalf serum, 100 units per ml penicillin, 100 micrograms per mlstreptomycin (Gibco/Life Technologies, Gaithersburg, Md.), 400 nM bovineinsulin (Sigma), 125 mM dexamethasone , 0.5 mM IBMX, and fungizone.Cells were routinely passaged by trypsinization and dilution when theyreached 90% confluence. Cells were seeded into 96-well plates(Falcon-Primaria #3872) at a density of 10000 cells/well for use inRT-PCR analysis.

[0258] For Northern blotting or other analyses, cells may be seeded onto100 mm or other standard tissue culture plates and treated similarly,using appropriate volumes of medium and oligonucleotide.

[0259] Treatment with Antisense Compounds:

[0260] When cells reached 70% confluency, they were treated witholigonucleotide. For cells grown in 96-well plates, wells were washedonce with 100 μL OPTI-MEM™-1 reduced-serum medium (InvitrogenCorporation, Carlsbad, Calif.) and then treated with 130 μL ofOPTI-MEM™-1 containing 3.75 μg/mL LIPOFECTIN™ (Invitrogen Corporation,Carlsbad, Calif.) and the desired concentration of oligonucleotide.After 4-7 hours of treatment, the medium was replaced with fresh medium.Cells were harvested 16-24 hours after oligonucleotide treatment.

[0261] The concentration of oligonucleotide used varies from cell lineto cell line. To determine the optimal oligonucleotide concentration fora particular cell line, the cells are treated with a positive controloligonucleotide at a range of concentrations. For human cells thepositive control oligonucleotide is selected from either ISIS 13920(TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 1) which is targeted to human H-ras,or ISIS 18078, (GTGCGCGCGAGCCCGAAATC, SEQ ID NO: 2) which is targeted tohuman Jun-N-terminal kinase-2 (JNK2). Both controls are2′-O-methoxyethyl gapmers (2′-O-methoxyethyls shown in bold) with aphosphorothioate backbone. For mouse or rat cells the positive controloligonucleotide is ISIS 15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 3, a2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown in bold) with aphosphorothioate backbone which is targeted to both mouse and rat c-raf.The concentration of positive control oligonucleotide that results in80% inhibition of c-Ha-ras (for ISIS 13920) or c-raf (for ISIS 15770)mRNA is then utilized as the screening concentration for newoligonucleotides in subsequent experiments for that cell line. If 80%inhibition is not achieved, the lowest concentration of positive controloligonucleotide that results in 60% inhibition of H-ras or c-raf mRNA isthen utilized as the oligonucleotide screening concentration insubsequent experiments for that cell line. If 60% inhibition is notachieved, that particular cell line is deemed as unsuitable foroligonucleotide transfection experiments. The concentrations ofantisense oligonucleotides used herein are from 50 nM to 300 nM.

Example 10 Analysis of Oligonucleotide Inhibition of Resistin Expression

[0262] Antisense modulation of resistin expression can be assayed in avariety of ways known in the art. For example, resistin mRNA levels canbe quantitated by, e.g., Northern blot analysis, competitive polymerasechain reaction (PCR), or real-time PCR (RT-PCR). Real-time quantitativePCR is presently preferred. RNA analysis can be performed on totalcellular RNA or poly(A)+ mRNA. The preferred method of RNA analysis ofthe present invention is the use of total cellular RNA as described inother examples herein. Methods of RNA isolation are taught in, forexample, Ausubel, F. M. et al., Current Protocols in Molecular Biology,Volume 1, pp. 4.1.1-4.2.9 and 4.5.1-4.5.3, John Wiley & Sons, Inc.,1993. Northern blot analysis is routine in the art and is taught in, forexample, Ausubel, F. M. et al., Current Protocols in Molecular Biology,Volume 1, pp. 4.2.1-4.2.9, John Wiley & Sons, Inc., 1996. Real-timequantitative (PCR) can be conveniently accomplished using thecommercially available ABI PRISM™ 7700 Sequence Detection System,available from PE-Applied Biosystems, Foster City, Calif. and usedaccording to manufacturer's instructions.

[0263] Protein levels of resistin can be quantitated in a variety ofways well known in the art, such as immunoprecipitation, Western blotanalysis (immunoblotting), ELISA or fluorescence-activated cell sorting(FACS). Antibodies directed to resistin can be identified and obtainedfrom a variety of sources, such as the MSRS catalog of antibodies (AerieCorporation, Birmingham, Mich.), or can be prepared via conventionalantibody generation methods. Methods for preparation of polyclonalantisera are taught in, for example, Ausubel, F. M. et al., (CurrentProtocols in Molecular Biology, Volume 2, pp. 11.12.1-11.12.9, JohnWiley & Sons, Inc., 1997). Preparation of monoclonal antibodies istaught in, for example, Ausubel, F. M. et al., (Current Protocols inMolecular Biology, Volume 2, pp. 11.4.1-11.11.5, John Wiley & Sons,Inc., 1997).

[0264] Immunoprecipitation methods are standard in the art and can befound at, for example, Ausubel, F. M. et al., (Current Protocols inMolecular Biology, Volume 2, pp. 10.16.1-10.16.11, John Wiley & Sons,Inc., 1998). Western blot (immunoblot) analysis is standard in the artand can be found at, for example, Ausubel, F. M. et al., (CurrentProtocols in Molecular Biology, Volume 2, pp. 10.8.1-10.8.21, John Wiley& Sons, Inc., 1997). Enzyme-linked immunosorbent assays (ELISA) arestandard in the art and can be found at, for example, Ausubel, F. M. etal., (Current Protocols in Molecular Biology, Volume 2, pp.11.2.1-11.2.22, John Wiley & Sons, Inc., 1991).

Example 11 Poly(A)+ mRNA Isolation

[0265] Poly(A)+ mRNA was isolated according to Miura et al., (Clin.Chem., 1996, 42, 1758-1764). Other methods for poly(A)+ mRNA isolationare taught in, for example, Ausubel, F. M. et al., (Current Protocols inMolecular Biology, Volume 1, pp. 4.5.1-4.5.3, John Wiley & Sons, Inc.,1993). Briefly, for cells grown on 96-well plates, growth medium wasremoved from the cells and each well was washed with 200 μL cold PBS. 60μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5%NP-40, 20 mM vanadyl-ribonucleoside complex) was added to each well, theplate was gently agitated and then incubated at room temperature forfive minutes. 55 μL of lysate was transferred to Oligo d(T) coated96-well plates (AGCT Inc., Irvine, Calif.). Plates were incubated for 60minutes at room temperature, washed 3 times with 200 μL of wash buffer(10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash,the plate was blotted on paper towels to remove excess wash buffer andthen air-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH7.6), preheated to 70° C., was added to each well, the plate wasincubated on a 90° C. hot plate for 5 minutes, and the eluate was thentransferred to a fresh 96-well plate.

[0266] Cells grown on 100 mm or other standard plates may be treatedsimilarly, using appropriate volumes of all solutions.

Example 12 Total RNA Isolation

[0267] Total RNA was isolated using an RNEASY 96™ kit and bufferspurchased from Qiagen Inc. (Valencia, Calif.) following themanufacturer's recommended procedures. Briefly, for cells grown on96-well plates, growth medium was removed from the cells and each wellwas washed with 200 μL cold PBS. 150 μL Buffer RLT was added to eachwell and the plate vigorously agitated for 20 seconds. 150 μL of 70%ethanol was then added to each well and the contents mixed by pipettingthree times up and down. The samples were then transferred to the RNEASY96™ well plate attached to a QIAVAC™ manifold fitted with a wastecollection tray and attached to a vacuum source. Vacuum was applied for1 minute. 500 μL of Buffer RW1 was added to each well of the RNEASY 96™plate and incubated for 15 minutes and the vacuum was again applied for1 minute. An additional 500 μL of Buffer RW1 was added to each well ofthe RNEASY 96™ plate and the vacuum was applied for 2 minutes. 1 mL ofBuffer RPE was then added to each well of the RNEASY 96™ plate and thevacuum applied for a period of 90 seconds. The Buffer RPE wash was thenrepeated and the vacuum was applied for an additional 3 minutes. Theplate was then removed from the QIAVAC™ manifold and blotted dry onpaper towels. The plate was then re-attached to the QIAVAC™ manifoldfitted with a collection tube rack containing 1.2 mL collection tubes.RNA was then eluted by pipetting 170 μL water into each well, incubating1 minute, and then applying the vacuum for 3 minutes.

[0268] The repetitive pipetting and elution steps may be automated usinga QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia, Calif.). Essentially,after lysing of the cells on the culture plate, the plate is transferredto the robot deck where the pipetting, DNase treatment and elution stepsare carried out.

Example 13 Real-Time Quantitative PCR Analysis of Resistin mRNA Levels

[0269] Quantitation of resistin MRNA levels was determined by real-timequantitative PCR using the ABI PRISM™ 7700 Sequence Detection System(PE-Applied Biosystems, Foster City, Calif.) according to manufacturer'sinstructions. This is a closed-tube, non-gel-based, fluorescencedetection system which allows high-throughput quantitation of polymerasechain reaction (PCR) products in real-time. As opposed to standard PCRin which amplification products are quantitated after the PCR iscompleted, products in real-time quantitative PCR are quantitated asthey accumulate. This is accomplished by including in the PCR reactionan oligonucleotide probe that anneals specifically between the forwardand reverse PCR primers, and contains two fluorescent dyes. A reporterdye (e.g., FAM or JOE, obtained from either PE-Applied Biosystems,Foster City, Calif., Operon Technologies Inc., Alameda, Calif. orIntegrated DNA Technologies Inc., Coralville, Iowa) is attached to the5′ end of the probe and a quencher dye (e.g., TAMRA, obtained fromeither PE-Applied Biosystems, Foster City, Calif., Operon TechnologiesInc., Alameda, Calif. or Integrated DNA Technologies Inc., Coralville,Iowa) is attached to the 3′ end of the probe. When the probe and dyesare intact, reporter dye emission is quenched by the proximity of the 3′quencher dye. During amplification, annealing of the probe to the targetsequence creates a substrate that can be cleaved by the 5′-exonucleaseactivity of Taq polymerase. During the extension phase of the PCRamplification cycle, cleavage of the probe by Taq polymerase releasesthe reporter dye from the remainder of the probe (and hence from thequencher moiety) and a sequence-specific fluorescent signal isgenerated. With each cycle, additional reporter dye molecules arecleaved from their respective probes, and the fluorescence intensity ismonitored at regular intervals by laser optics built into the ABI PRISM™7700 Sequence Detection System. In each assay, a series of parallelreactions containing serial dilutions of mRNA from untreated controlsamples generates a standard curve that is used to quantitate thepercent inhibition after antisense oligonucleotide treatment of testsamples.

[0270] Prior to quantitative PCR analysis, primer-probe sets specific tothe target gene being measured are evaluated for their ability to be“multiplexed” with a GAPDH amplification reaction. In multiplexing, boththe target gene and the internal standard gene GAPDH are amplifiedconcurrently in a single sample. In this analysis, mRNA isolated fromuntreated cells is serially diluted. Each dilution is amplified in thepresence of primer-probe sets specific for GAPDH only, target gene only(“single-plexing”), or both (multiplexing). Following PCR amplification,standard curves of GAPDH and target mRNA signal as a function ofdilution are generated from both the single-plexed and multiplexedsamples. If both the slope and correlation coefficient of the GAPDH andtarget signals generated from the multiplexed samples fall within 10% oftheir corresponding values generated from the single-plexed samples, theprimer-probe set specific for that target is deemed multiplexable. Othermethods of PCR are also known in the art.

[0271] PCR reagents were obtained from Invitrogen Corporation,(Carlsbad, Calif.). RT-PCR reactions were carried out by adding 20 μLPCR cocktail (2.5× PCR buffer (−MgC12), 6.6 mM MgCl2, 375 μM each ofdATP, dCTP, dCTP and dGTP, 375 nM each of forward primer and reverseprimer, 125 nM of probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM®Taq, 5 Units MuLV reverse transcriptase, and 2.5× ROX dye) to 96-wellplates containing 30 μL total RNA solution. The RT reaction was carriedout by incubation for 30 minutes at 48° C. Following a 10 minuteincubation at 95° C. to activate the PLATINUM® Taq, 40 cycles of atwo-step PCR protocol were carried out: 95° C. for 15 seconds(denaturation) followed by 60° C. for 1.5 minutes (annealing/extension).

[0272] Gene target quantities obtained by real time RT-PCR arenormalized using either the expression level of GAPDH, a gene whoseexpression is constant, or by quantifying total RNA using RiboGreen™(Molecular Probes, Inc. Eugene, Oreg.). GAPDH expression is quantifiedby real time RT-PCR, by being run simultaneously with the target,multiplexing, or separately. Total RNA is quantified using RiboGreenTMRNA quantification reagent from Molecular Probes. Methods of RNAquantification by RiboGreenTM are taught in Jones, L. J., et al,(Analytical Biochemistry, 1998, 265, 368-374).

[0273] In this assay, 170 μL of RiboGreenTM working reagent (RiboGreenTMreagent diluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipettedinto a 96-well plate containing 30 μL purified, cellular RNA. The plateis read in a CytoFluor 4000 (PE Applied Biosystems) with excitation at480 nm and emission at 520 nm.

[0274] Probes and primers to human resistin were designed to hybridizeto a human resistin sequence, using published sequence information(GenBank accession number NM_(—)020415.1, incorporated herein as SEQ IDNO:4). For human resistin the PCR primers were:

[0275] forward primer: AAGCCATCAATGAGAGGATCCA (SEQ ID NO: 5)

[0276] reverse primer: TCCAGGCCAATGCTGCTTA (SEQ ID NO: 6) and the

[0277] PCR probe was: FAM-TCGCCGGCTCCCTAATATTTAGGGCA-TAMRA (SEQ ID NO:7) where FAM is the fluorescent dye and TAMRA is the quencher dye. Forhuman GAPDH the PCR primers were:

[0278] forward primer: GAAGGTGAAGGTCGGAGTC(SEQ ID NO:8)

[0279] reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO:9) and the

[0280] PCR probe was: 5′ JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3′ (SEQ ID NO:10) where JOE is the fluorescent reporter dye and TAMRA is the quencherdye.

[0281] Probes and primers to mouse resistin were designed to hybridizeto a mouse resistin sequence, using published sequence information(GenBank accession number 1783_(—)080, incorporated herein as SEQ IDNO:11). For mouse resistin the PCR primers were:

[0282] forward primer: TCGTGGGACATTCGTGAAGA (SEQ ID NO:12)

[0283] reverse primer: CGGGCTGCTGTCCAGTCT (SEQ ID NO: 13) and the

[0284] PCR probe was: FAM-AAGTGTGTCACTGCCAGTGTGCAAGGA-TAMRA (SEQ ID NO:14) where FAM is the fluorescent reporter dye and TAMRA is the quencherdye. For mouse GAPDH the PCR primers were:

[0285] forward primer: GGCAAATTCAACGGCACAGT(SEQ ID NO:15)

[0286] reverse primer: GGGTCTCGCTCCTGGAAGAT(SEQ ID NO:16) and the

[0287] PCR probe was: 5′ JOE-AAGGCCGAGAATGGGAAGCTTGTCATC-TAMRA 3′ (SEQID NO: 17) where JOE is the fluorescent reporter dye and TAMRA is thequencher dye.

Example 14 Northern Blot Analysis of Resistin mRNA Levels

[0288] Eighteen hours after antisense treatment, cell monolayers werewashed twice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST “B” Inc.,Friendswood, Tex.). Total RNA was prepared following manufacturer'srecommended protocols. Twenty micrograms of total RNA was fractionatedby electrophoresis through 1.2% agarose gels containing 1.1%formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNAwas transferred from the gel to HYBOND™-N+ nylon membranes (AmershamPharmacia Biotech, Piscataway, N.J.) by overnight capillary transferusing a Northern/Southern Transfer buffer system (TEL-TEST “B” Inc.,Friendswood, Tex.). RNA transfer was confirmed by UV visualization.Membranes were fixed by UV cross-linking using a STRATALINKER™ UVCrosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then probedusing QUICKHYB™ hybridization solution (Stratagene, La Jolla, Calif.)using manufacturer's recommendations for stringent conditions.

[0289] To detect human resistin, a human resistin specific probe wasprepared by PCR using the forward primer AAGCCATCAATGAGAGGATCCA (SEQ IDNO: 5) and the reverse primer TCCAGGCCAATGCTGCTTA (SEQ ID NO: 6). Tonormalize for variations in loading and transfer efficiency membraneswere stripped and probed for human glyceraldehyde-3-phosphatedehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.).

[0290] To detect mouse resistin, a mouse resistin specific probe wasprepared by PCR using the forward primer TCGTGGGACATTCGTGAAGA (SEQ IDNO: 12) and the reverse primer CGGGCTGCTGTCCAGTCT (SEQ ID NO: 13). Tonormalize for variations in loading and transfer efficiency membraneswere stripped and probed for mouse glyceraldehyde-3-phosphatedehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.).

[0291] Hybridized membranes were visualized and quantitated using aPHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3 (Molecular Dynamics,Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreatedcontrols.

Example 15 Antisense Inhibition of Human Resistin Expression by ChimericPhosphorothioate Oligonucleotides having 2′-MOE Wings and a Deoxy Gap

[0292] In accordance with the present invention, a series ofoligonucleotides were designed to target different regions of the humanresistin RNA, using published sequences (GenBank accession numberNM_(—)020415.1, incorporated herein as SEQ ID NO: 4, and the complementof residues 116001-118000 of GenBank accession number AC008763.5,incorporated herein as SEQ ID NO: 18). The oligonucleotides are shown inTable 1. “Target site” indicates the first (5′-most) nucleotide numberon the particular target sequence to which the oligonucleotide binds.All compounds in Table 1 are chimeric oligonucleotides (“gapmers”) 20nucleotides in length, composed of a central “gap” region consisting often 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′directions) by five-nucleotide “wings”. The wings are composed of2′-methoxyethyl (2′-MOE)nucleotides. The internucleoside (backbone)linkages are phosphorothioate (P═S) throughout the oligonucleotide. Allcytidine residues are 5-methylcytidines. The compounds were analyzed fortheir effect on human resistin mRNA levels by quantitative real-time PCRas described in other examples herein. Data are averages from twoexperiments in which Jurkat cells were treated with the oligonucleotidesof the present invention. If present, “N.D.” indicates “no data”. TABLE1 Inhibition of human resistin mPNA levels by chimeric phosphorothioateoligonucleotides having 2′-MOE wings and a deoxy gap REGION TARGET SEQID TARGET SEQ ID ISIS # REGION NO SITE SEQUENCE % INHIB NO 165438 Start4 17 gagagctttcatcctgcagg 55 19 Codon 165440 Start 4 27ggaggagacagagagctttc 62 20 Codon 165442 Coding 4 60 tgctagacaccaacagcccc0 21 165444 Coding 4 66 gggtcttgctagacaccaac 0 22 165763 Coding 4 73gagcacagggtcttgctaga 0 23 165765 Coding 4 79 tccatggagcacagggtctt 3 24165767 Coding 4 91 ttgatggcttcttccatgga 0 25 165769 Coding 4 97ctctcattgatggcttcttc 55 26 165771 Coding 4 108 cctcctggatcctctcattg 8127 165773 Coding 4 116 gccggcgacctcctggatcc 91 28 165775 Coding 4 122tagggagccggcgacctcct 58 29 165777 Coding 4 125 tattagggagccggcgacct 5530 165780 Coding 4 140 gcttattgccctaaatatta 30 31 165782 Coding 4 151aggccaatgctgcttattgc 96 32 165784 Coding 4 154 tccaggccaatgctgcttat 8833 165786 Coding 4 159 ggcactccaggccaatgctg 81 34 165788 Coding 4 226caagtgcagccggtgacggc 0 35 165790 Coding 4 233 ggagccacaagtgcagccgg 28 36165792 Coding 4 239 acaggcggagccacaagtgc 13 37 165794 Coding 4 255gcacatcccacgagccacag 57 38 165796 Coding 4 279 ggcagtgacatgtggtctcg 4139 165798 Coding 4 286 gcgcactggcagtgacatgt 47 40 165800 Coding 4 291tgcccgcgcactggcagtga 61 41 165802 Coding 4 301 gtccagtccatgcccgcgca 1142 165804 Coding 4 328 ggctgcacacgacagcagcg 0 43 165806 Stop 4 340gcgcgacctcagggctgcac 83 44 Codon 165808 3′UTR 4 389 aacccctccggacctggagc57 45 165810 3′UTR 4 407 tatttccagctcccccgcaa 12 46 165812 3′UTR 4 412aggtttatttccagctcccc 54 47 165814 3′UTR 4 418 atctccaggtttatttccag 64 48165816 3′UTR 4 423 tcatcatctccaggtttatt 74 49 165818 3′UTR 4 428catcatcatcatctccaggt 88 50 165820 Exon: 18 369 ggtcctcacttagggagccg 2051 Intron Junction 165830 Intron: 18 1142 ggcgaagcctgcagcccgga 0 52 ExonJunction

[0293] As shown in Table 1, SEQ ID NOs. 19, 20, 26, 27, 28, 29, 30, 32,33, 34, 38, 41, 44, 45, 47, 48, 49 and 50 demonstrated at least 50%inhibition of human resistin expression in this assay and are thereforepreferred. The target sites to which these preferred sequences arecomplementary are herein referred to as “preferred target regions” andare therefore preferred sites for targeting by compounds of the presentinvention. These preferred target regions are shown in Table 3. Thesequences represent the reverse complement of the preferred antisensecompounds shown in Table 1. “Target site” indicates the first (5′-most)nucleotide number of the corresponding target nucleic acid. Also shownin Table 3 is the species in which each of the preferred target regionswas found.

Example 16 Antisense Inhibition of Mouse Resistin Expression by ChimericPhosphorothioate Oligonucleotides having 2′-MOE Wings and a Deoxy Gap

[0294] In accordance with the present invention, a second series ofoligonucleotides were designed to target different regions of the mouseresistin RNA, using published sequences (the sequence presented in FIG.1: Steppan et al., Nature, 2001, 409, 307-312, incorporated herein asSEQ ID NO: 11, and an assembly of contigs from GenBank accession numberAC079491.1, incorporated herein as SEQ ID NO: 53). The oligonucleotidesare shown in Table 2. “Target site” indicates the first (5′-most)nucleotide number on the particular target sequence to which theoligonucleotide binds. All compounds in Table 2 are chimericoligonucleotides (“gapmers”) 20 nucleotides in length, composed of acentral “gap” region consisting of ten 2′-deoxynucleotides, which isflanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”.The wings are composed of 2′-methoxyethyl (2′-MOE)nucleotides. Theinternucleoside (backbone) linkages are phosphorothioate (P═S)throughout the oligonucleotide. All cytidine residues are5-methylcytidines. The compounds were analyzed for their effect on mouseresistin mRNA levels by quantitative real-time PCR as described in otherexamples herein. Data are averages from two experiments in whichdifferentiated 3T3-L1 cells were treated with the oligonucleotides ofthe present invention. The positive control for each datapoint isidentified in the table by sequence ID number. If present, “N.D.”indicates “no data”. TABLE 2 Inhibition of mouse resistin mENA levels bychimeric phosphorothioate oligonucleotides having 2′-MOE wings and adeoxy gap TARGET CONTROL SEQ ID TARGE SEQ ID SEQ ID ISIS # REGION NOSITE SEQUENCE % INHIB NO NO 165359 5′UTR 11 14 attagctcctgtcccacagc 3654 1 165360 5′UTR 11 18 gggtattagctcctgtccca 39 55 1 165361 5′UTR 11 28actcagttctgggtattagc 21 56 1 165362 5′UTR 11 41 ttagcaggacacaactcagt 657 1 165363 5′UTR 11 47 gaggacttagcaggacacaa 14 58 1 165364 5′UTR 11 51ggcagaggacttagcaggac 9 59 1 165365 5′UTR 11 60 tgggtacgtggcagaggact 4060 1 165366 Start 11 68 tcatcccgtgggtacgtggc 5 61 1 Codon 165367 Start11 76 aaggttcttcatcccgtggg 19 62 1 Codon 165368 Start 11 84ggaaatgaaaggttcttcat 4 63 1 Codon 165369 Coding 11 116gttcagggacaaggaagaaa 17 64 1 165370 Coding 11 125 agcccagcagttcagggaca34 65 1 165371 Coding 11 129 ctggagcccagcagttcagg 0 66 1 165372 Coding11 134 gcatgctggagcccagCagt 3 67 1 165373 Coding 11 139cagtggcatgctggagccca 15 68 1 165374 Coding 11 151 atcgatgggacacagtggca52 69 1 165375 Coding 11 155 cttcatcgatgggacacagt 8 70 1 165376 Coding11 162 tcgatggcttcatcgatggg 0 71 1 165377 Coding 11 172gatcttcttgtcgatggctt 26 72 1 165378 Coding 11 191 gggagttgaagtcttgtttg 873 1 165379 Coding 11 194 acagggagttgaagtcttgt 21 74 1 165380 Coding 11197 gaaacagggagttgaagtct 14 75 1 165381 Coding 11 204gcatttggaaacagggagtt 30 76 1 165382 Coding 11 209 ttattgcatttggaaacagg 077 1 165383 Coding 11 211 ctttattgcatttggaaaca 20 78 1 165384 Coding 11220 gccaatgttctttattgcat 35 79 1 165385 Coding 11 228caatttaagccaatgttctt 8 80 1 165386 Coding 11 229 gcaatttaagccaatgttct 081 1 165387 Coding 11 235 tgtccagcaatttaagccaa 46 82 1 165388 Coding 11240 gagactgtccagcaatttaa 52 83 1 165389 Coding 11 252ttccctctggaggagactgt 0 84 1 165390 Coding 11 258 gccaacttccctctggagga 6685 1 165391 Coding 11 259 ggccaacttccctctggagg 38 86 1 165392 Coding 11264 caggaggccaacttccctct 24 87 1 165393 Coding 11 267gggcaggaggccaacttccc 11 88 1 165394 Coding 11 282 actgctgtgccttctgggca 089 1 165395 Coding 11 299 cacaggagcagctcaagact 3 90 1 165396 Coding 11322 gtcccacgagccacaggcag 12 91 1 165397 Coding 11 324atgtcccacgagccacaggc 25 92 1 165398 Coding 11 332 cttcacgaatgtcccacgag37 93 1 165399 Coding 11 338 ctttttcttcacgaatgtcc 25 94 1 165400 Coding11 354 cactggcagtgacacacttt 61 95 1 165401 Coding 11 358tgcacactggcagtgacaca 67 96 1 165402 Coding 11 363 atccttgcacactggcagtg 897 1 165403 Coding 11 367 gtctatccttgcacactggc 70 98 1 165404 Coding 11373 tgtccagtctatccttgcac 58 99 1 165405 Coding 11 377ctgctgtccagtctatcctt 66 100 1 165406 Coding 11 384 cagcgggctgctgtccagtc69 101 1 165407 Coding 11 388 acagcagcgggctgctgtcc 40 102 1 165408Coding 11 398 cctgcagcttacagcagcgg 3 103 1 165409 Coding 11 401cgacctgcagcttacagcag 0 104 1 165410 Coding 11 406 ggaagcgacctgcagcttac 2105 1 165411 Stop 11 409 tcaggaagcgacctgcagct 0 106 1 Codon 165412 Stop11 418 tccccgacatcaggaagcga 10 107 1 Codon 165413 Stop 11 424ctcacttccccgacatcagg 0 108 1 Codon 165414 3′UTR 11 437gctggaaaccacgctcactt 50 109 1 165415 3′UTR 11 441 ctgtgctggaaaccacgctc 6110 1 165416 3′UTR 11 456 tacaggaacgggtggctgtg 0 111 1 165417 3′UTR 11468 catctctggagctacaggaa 18 112 1 165418 3′UTR 11 470gacatctctggaqctacagg 23 113 1 165419 3′UTR 11 474 atcagacatctctggagcta22 114 1 165420 3′UTR 11 479 aggacatcagacatctctgg 24 115 1 165421 3′UTR11 486 agaccggaggacatcagaca 26 116 1 165422 3′UTR 11 529tgcttgtgtgtggattcgcg 62 117 1 165423 3′UTR 11 531 tgtgcttgtgtgtggattcg78 118 1 165424 Exon: 53 8737 tatcacttaccgtggcagag 10 119 1 IntronJunction 165425 Intron 53 8796 atggatggacattcagcagc 31 120 1 165426Intron: 53 8899 cccgtgggtactgcaagaga 23 121 1 Exon Junction 165427Intron 53 9350 acgttcacggtaaaatattt 1 122 1 165428 Intron 53 9486gattctgtctcaacaaagca 42 123 1 165429 Intron 53 9630 cccagaacccatttagaagc32 124 1 165430 Intron: 53 10143 actgctgtgcctgcaaggaa 6 125 1 ExonJunction 165431 Exon: 53 10285 ctcaactgaccgacatcagg 10 126 1 IntronJunction 165432 Intron 53 10307 cataagaaggacaagctcag 0 127 1 165433Intron 53 11957 ctccatggcttctgcatcag 33 128 1 165434 Intron 53 12110cataagatccactacagggc 21 129 1 165435 Intron 53 12692taaccattgacccatctttc 0 130 1 165436 Intron: 53 12724ctcacttcccctgaaaaagc 5 131 1 Exon Junction

[0295] As shown in Table 2, SEQ ID NOs 60, 69, 82, 83, 85, 95, 96, 98,99, 100, 101, 102, 109, 117, 118 and 123 demonstrated at least 40%inhibition of mouse resistin expression in this experiment and aretherefore preferred. The target sites to which these preferred sequencesare complementary are herein referred to as “preferred target regions”and are therefore preferred sites for targeting by compounds of thepresent invention. These preferred target regions are shown in Table 3.The sequences represent the reverse complement of the preferredantisense compounds shown in Table 1. “Target site” indicates the first(5′-most) nucleotide number of the corresponding target nucleic acid.Also shown in Table 3 is the species in which each of the preferredtarget regions was found. TABLE 3 Sequence and position of preferredtarget regions identified in resistin. TARGET SEQ ID TARGET REV COMP SEQID SITEID NO SITE SEQUENCE OF SEQ ID ACTIVE IN NO 80897 4 17cctgcaggatgaaagctctc 19 H. sapiens 132 80899 4 27 gaaagctctctgtctcctcc20 H. sapiens 133 80911 4 97 gaagaagccatcaatgagag 26 H. sapiens 13480913 4 108 caatgagaggatccaggagg 27 H. sapiens 135 80915 4 116ggatccaggaggtcgccggc 28 H. sapiens 136 80917 4 122 aggaggtcgccggctcccta29 H. sapiens 137 80919 4 125 aggtcgccggctccctaata 30 H. sapiens 13880924 4 151 gcaataagcagcattggcct 32 H. sapiens 139 80926 4 154ataagcagcattggcctgga 33 H. sapiens 140 80928 4 159 cagcattggcctggagtgcc34 H. sapiens 141 80936 4 255 ctgtggctcgtgggatgtgc 38 H. sapiens 14280942 4 291 tcactgccagtgcgcgggca 41 H. sapiens 143 80948 4 340gtgcagccctgaggtcgcgc 44 H. sapiens 144 80950 4 389 gctccaggtccggaggggtt45 H. sapiens 145 80954 4 412 ggggagctggaaataaacct 47 H. sapiens 14680956 4 418 ctggaaataaacctggagat 48 H. sapiens 147 80958 4 423aataaacctggagatgatga 49 H. sapiens 148 80960 4 428 acctggagatgatgatgatg50 H. sapiens 149 80824 11 60 agtcctctgccacgtaccca 60 M. musculus 15080833 11 151 tgccactgtgtcccatcgat 69 M. musculus 151 80846 11 235ttggcttaaattgctggaca 82 M. musculus 152 80847 11 240ttaaattgctggacagtctc 83 M. musculus 153 80849 11 258tcctccagagggaagttggc 85 M. musculus 154 80859 11 354aaagtgtgtcactgccagtg 95 M. musculus 155 80860 11 358tgtgtcactgccagtgtgca 96 M. musculus 156 80862 11 367gccagtgtgcaaggatagac 98 M. musculus 157 80863 11 373gtgcaaggatagactggaca 99 M. musculus 158 80864 11 377aaggatagactggacagcag 100 M. musculus 159 80865 11 384gactggacagcagcccgctg 101 M. musculus 160 80866 11 388ggacagcagcccgctgctgt 102 M. musculus 161 80873 11 437aagtgagcgtggtttccagc 109 M. musculus 162 80881 11 529cgcgaatccacacacaagca 117 M. musculus 163 80882 11 531cgaatccacacacaagcaca 118 M. musculus 164 80887 53 9486tgctttgttgagacagaatc 123 M. muscuius 165

[0296] As these “preferred target regions” have been found byexperimentation to be open to, and accessible for, hybridization withthe antisense compounds of the present invention, one of skill in theart will recognize or be able to ascertain, using no more than routineexperimentation, further embodiments of the invention that encompassother compounds that specifically hybridize to these sites andconsequently inhibit the expression of resistin.

[0297] In one embodiment, the “preferred target region” may be employedin screening candidate antisense compounds. “Candidate antisensecompounds” are those that inhibit the expression of a nucleic acidmolecule encoding resistin and which comprise at least an 8-nucleobaseportion which is complementary to a preferred target region. The methodcomprises the steps of contacting a preferred target region of a nucleicacid molecule encoding resistin with one or more candidate antisensecompounds, and selecting for one or more candidate antisense compoundswhich inhibit the expression of a nucleic acid molecule encodingresistin. Once it is shown that the candidate antisense compound orcompounds are capable of inhibiting the expression of a nucleic acidmolecule encoding resistin, the candidate antisense compound may beemployed as an antisense compound in accordance with the presentinvention.

[0298] According to the present invention, antisense compounds includeribozymes, external guide sequence (EGS) oligonucleotides (oligozymes),and other short catalytic RNAs or catalytic oligonucleotides whichhybridize to the target nucleic acid and modulate its expression.

Example 17 Western Blot Analysis of Resistin Protein Levels

[0299] Western blot analysis (immunoblot analysis) is carried out usingstandard methods. Cells are harvested 16-20 h after oligonucleotidetreatment, washed once with PBS, suspended in Laemmli buffer (100ul/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gelsare run for 1.5 hours at 150 V, and transferred to membrane for westernblotting. Appropriate primary antibody directed to resistin is used,with a radiolabeled or fluorescently labeled secondary antibody directedagainst the primary antibody species. Bands are visualized using aPHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale Calif.).

1 165 1 20 DNA Artificial Sequence Antisense Oligonucleotide 1tccgtcatcg ctcctcaggg 20 2 20 DNA Artificial Sequence AntisenseOligonucleotide 2 gtgcgcgcga gcccgaaatc 20 3 20 DNA Artificial SequenceAntisense Oligonucleotide 3 atgcattctg cccccaagga 20 4 457 DNA H.sapiens CDS (25)...(351) 4 agcccaccga gaggcgcctg cagg atg aaa gct ctctgt ctc ctc ctc ctc 51 Met Lys Ala Leu Cys Leu Leu Leu Leu 1 5 cct gtcctg ggg ctg ttg gtg tct agc aag acc ctg tgc tcc atg gaa 99 Pro Val LeuGly Leu Leu Val Ser Ser Lys Thr Leu Cys Ser Met Glu 10 15 20 25 gaa gccatc aat gag agg atc cag gag gtc gcc ggc tcc cta ata ttt 147 Glu Ala IleAsn Glu Arg Ile Gln Glu Val Ala Gly Ser Leu Ile Phe 30 35 40 agg gca ataagc agc att ggc ctg gag tgc cag agc gtc acc tcc agg 195 Arg Ala Ile SerSer Ile Gly Leu Glu Cys Gln Ser Val Thr Ser Arg 45 50 55 ggg gac ctg gctact tgc ccc cga ggc ttc gcc gtc acc ggc tgc act 243 Gly Asp Leu Ala ThrCys Pro Arg Gly Phe Ala Val Thr Gly Cys Thr 60 65 70 tgt ggc tcc gcc tgtggc tcg tgg gat gtg cgc gcc gag acc aca tgt 291 Cys Gly Ser Ala Cys GlySer Trp Asp Val Arg Ala Glu Thr Thr Cys 75 80 85 cac tgc cag tgc gcg ggcatg gac tgg acc gga gcg cgc tgc tgt cgt 339 His Cys Gln Cys Ala Gly MetAsp Trp Thr Gly Ala Arg Cys Cys Arg 90 95 100 105 gtg cag ccc tgaggtcgcgcgc agcgcgtgca cagcgcgggc ggaggcggct 391 ccaggtccgg aggggttgcgggggagctgg aaataaacct ggagatgatg atgatgatga 451 tgatgg 457 5 22 DNAArtificial Sequence PCR Primer 5 aagccatcaa tgagaggatc ca 22 6 19 DNAArtificial Sequence PCR Primer 6 tccaggccaa tgctgctta 19 7 26 DNAArtificial Sequence PCR Probe 7 tcgccggctc cctaatattt agggca 26 8 19 DNAArtificial Sequence PCR Primer 8 gaaggtgaag gtcggagtc 19 9 20 DNAArtificial Sequence PCR Primer 9 gaagatggtg atgggatttc 20 10 20 DNAArtificial Sequence PCR Probe 10 caagcttccc gttctcagcc 20 11 591 DNA M.musculus CDS (84)...(428) 11 tcaacaagaa ggagctgtgg gacaggagct aatacccagaactgagttgt gtcctgctaa 60 gtcctctgcc acgtacccac ggg atg aag aac ctt tcattt ccc ctc ctt ttc 113 Met Lys Asn Leu Ser Phe Pro Leu Leu Phe 1 5 10ctt ttc ttc ctt gtc cct gaa ctg ctg ggc tcc agc atg cca ctg tgt 161 LeuPhe Phe Leu Val Pro Glu Leu Leu Gly Ser Ser Met Pro Leu Cys 15 20 25 cccatc gat gaa gcc atc gac aag aag atc aaa caa gac ttc aac tcc 209 Pro IleAsp Glu Ala Ile Asp Lys Lys Ile Lys Gln Asp Phe Asn Ser 30 35 40 ctg tttcca aat gca ata aag aac att ggc tta aat tgc tgg aca gtc 257 Leu Phe ProAsn Ala Ile Lys Asn Ile Gly Leu Asn Cys Trp Thr Val 45 50 55 tcc tcc agaggg aag ttg gcc tcc tgc cca gaa ggc aca gca gtc ttg 305 Ser Ser Arg GlyLys Leu Ala Ser Cys Pro Glu Gly Thr Ala Val Leu 60 65 70 agc tgc tcc tgtggc tct gcc tgt ggc tcg tgg gac att cgt gaa gaa 353 Ser Cys Ser Cys GlySer Ala Cys Gly Ser Trp Asp Ile Arg Glu Glu 75 80 85 90 aaa gtg tgt cactgc cag tgt gca agg ata gac tgg aca gca gcc cgc 401 Lys Val Cys His CysGln Cys Ala Arg Ile Asp Trp Thr Ala Ala Arg 95 100 105 tgc tgt aag ctgcag gtc gct tcc tga tgtcggggaa gtgagcgtgg 448 Cys Cys Lys Leu Gln ValAla Ser 110 tttccagcac agccacccgt tcctgtagct ccagagatgt ctgatgtcctccggtctcta 508 caggcacctg cactcacgtg cgcgaatcca cacacaagca cacatacttaaaaataaaac 568 aaaacaggct ggaaaaaaaa aaa 591 12 20 DNA ArtificialSequence PCR Primer 12 tcgtgggaca ttcgtgaaga 20 13 18 DNA ArtificialSequence PCR Primer 13 cgggctgctg tccagtct 18 14 27 DNA ArtificialSequence PCR Probe 14 aagtgtgtca ctgccagtgt gcaagga 27 15 20 DNAArtificial Sequence PCR Primer 15 ggcaaattca acggcacagt 20 16 20 DNAArtificial Sequence PCR Primer 16 gggtctcgct cctggaagat 20 17 27 DNAArtificial Sequence PCR Probe 17 aaggccgaga atgggaagct tgtcatc 27 182000 DNA H. sapiens 18 gagctgcggt gcaggaattc gtgtgccgga tttggttagctgagcccacc gagagggtaa 60 gtgacagctg ctcctgcgct tgccatggca ccagcggggaggctggggtc aaggctgagc 120 ctccatccct gtcccccaca tggggggaca ggggtccaggtccaggggca gatcctactc 180 cctccatggg ccggatcttc cccacagggc agggctgatccagctgtggg tctcttggtt 240 ccctctttca gcgcctgcag gatgaaagct ctctgtctcctcctcctccc tgtcctgggg 300 ctgttggtgt ctagcaagac cctgtgctcc atggaagaagccatcaatga gaggatccag 360 gaggtcgccg gctccctaag tgaggacccc ccacttgggcaagctcccca agggtctcag 420 agacctcact gatccctggc acagacctga ctccaacccagccccagcgc tcaccaaatc 480 tcatcctcaa atccaaccag atcataaatt caaccccaactccactccca acccctccga 540 ctgtccccac cttatccacg gctccaaacc caatccccgctctcactcca aaccttccct 600 tactccaaaa cacccaactc aagacagggt cctggaggccagtgagctcc tatgcccaca 660 gggacctagc tccaaaccaa cagggctagg ggaggatgggggagggaccg tttggtctca 720 cagctccccc tgtctccttt cctcctgccc cccagtatttagggcaataa gcagcattgg 780 cctggagtgc cagagcgtca cctccagggg ggacctggctacttgccccc gaggtgagtg 840 caggagactg ttgtccaggc gcccatttct gttccaagtcccctgggaat gccccctccc 900 cgccacgttc cccgtgtcca gcctctactc ccctaggatcttggtcctga ctcccagcct 960 tctccgccca ccatctggac actggtgtcc accctcactccctgcctcca gtgcccattc 1020 agtggttgga gcctccagcc gtccccgtcc ccacccccgcccccccaacc cccctccgcg 1080 ctccccaccc ccctcccgct cccaccctca gcctcccagctcagagtcca cgctcctgtg 1140 ttccgggctg caggcttcgc cgtcaccggc tgcacttgtggctccgcctg tggctcgtgg 1200 gatgtgcgcg ccgagaccac atgtcactgc cagtgcgcgggcatggactg gaccggagcg 1260 cgctgctgtc gtgtgcagcc ctgaggtcgc gcgcagcgcgtgcacagcgc gggcggaggc 1320 ggctccaggt ccggaggggt tgcgggggag ctggaaataaacctggagat gatgatgatg 1380 atgatgatga tgatgatgga gcggatctga gccctgcgtggtttctttag taggcccgga 1440 gggactgatc tagcgtctcc aagagagtgg ggcgcgtagctgctggaggg ggcggggaca 1500 ccgcgacttt ctactgcccc atcgcccttc ctcctatggggtctccaact gcttcctccg 1560 aaaatagggc ctgaacttcc tctagtgacg tccccacccaaggctcatgg ctgccttcaa 1620 gaggtgacgt ctcatctttg aggctacctt gacgctcaccctggggtctc cgacctcccc 1680 aggaagtggc tgggtccttt tcccccagtc ctcataatgaggcttcatcg aggacctggg 1740 tcagtctggg cagtggacgg gaccctccag ggccccaagactccaggagc cccaggtcag 1800 ggtggaaccc tgaatcatgt ctcagcccag agctggaacctgtacccctc acttcctacc 1860 tgcaaggagg aatccccaag gcacaggcaa agttgggttacggagagtca gggacgccta 1920 cctgacgtca cgcatcatca caagctcacg ttttcacacaggcaagtgca gttgtgagta 1980 gttagttaca accagataca 2000 19 20 DNAArtificial Sequence Antisense Oligonucleotide 19 gagagctttc atcctgcagg20 20 20 DNA Artificial Sequence Antisense Oligonucleotide 20 ggaggagacagagagctttc 20 21 20 DNA Artificial Sequence Antisense Oligonucleotide 21tgctagacac caacagcccc 20 22 20 DNA Artificial Sequence AntisenseOligonucleotide 22 gggtcttgct agacaccaac 20 23 20 DNA ArtificialSequence Antisense Oligonucleotide 23 gagcacaggg tcttgctaga 20 24 20 DNAArtificial Sequence Antisense Oligonucleotide 24 tccatggagc acagggtctt20 25 20 DNA Artificial Sequence Antisense Oligonucleotide 25 ttgatggcttcttccatgga 20 26 20 DNA Artificial Sequence Antisense Oligonucleotide 26ctctcattga tggcttcttc 20 27 20 DNA Artificial Sequence AntisenseOligonucleotide 27 cctcctggat cctctcattg 20 28 20 DNA ArtificialSequence Antisense Oligonucleotide 28 gccggcgacc tcctggatcc 20 29 20 DNAArtificial Sequence Antisense Oligonucleotide 29 tagggagccg gcgacctcct20 30 20 DNA Artificial Sequence Antisense Oligonucleotide 30 tattagggagccggcgacct 20 31 20 DNA Artificial Sequence Antisense Oligonucleotide 31gcttattgcc ctaaatatta 20 32 20 DNA Artificial Sequence AntisenseOligonucleotide 32 aggccaatgc tgcttattgc 20 33 20 DNA ArtificialSequence Antisense Oligonucleotide 33 tccaggccaa tgctgcttat 20 34 20 DNAArtificial Sequence Antisense Oligonucleotide 34 ggcactccag gccaatgctg20 35 20 DNA Artificial Sequence Antisense Oligonucleotide 35 caagtgcagccggtgacggc 20 36 20 DNA Artificial Sequence Antisense Oligonucleotide 36ggagccacaa gtgcagccgg 20 37 20 DNA Artificial Sequence AntisenseOligonucleotide 37 acaggcggag ccacaagtgc 20 38 20 DNA ArtificialSequence Antisense Oligonucleotide 38 gcacatccca cgagccacag 20 39 20 DNAArtificial Sequence Antisense Oligonucleotide 39 ggcagtgaca tgtggtctcg20 40 20 DNA Artificial Sequence Antisense Oligonucleotide 40 gcgcactggcagtgacatgt 20 41 20 DNA Artificial Sequence Antisense Oligonucleotide 41tgcccgcgca ctggcagtga 20 42 20 DNA Artificial Sequence AntisenseOligonucleotide 42 gtccagtcca tgcccgcgca 20 43 20 DNA ArtificialSequence Antisense Oligonucleotide 43 ggctgcacac gacagcagcg 20 44 20 DNAArtificial Sequence Antisense Oligonucleotide 44 gcgcgacctc agggctgcac20 45 20 DNA Artificial Sequence Antisense Oligonucleotide 45 aacccctccggacctggagc 20 46 20 DNA Artificial Sequence Antisense Oligonucleotide 46tatttccagc tcccccgcaa 20 47 20 DNA Artificial Sequence AntisenseOligonucleotide 47 aggtttattt ccagctcccc 20 48 20 DNA ArtificialSequence Antisense Oligonucleotide 48 atctccaggt ttatttccag 20 49 20 DNAArtificial Sequence Antisense Oligonucleotide 49 tcatcatctc caggtttatt20 50 20 DNA Artificial Sequence Antisense Oligonucleotide 50 catcatcatcatctccaggt 20 51 20 DNA Artificial Sequence Antisense Oligonucleotide 51ggtcctcact tagggagccg 20 52 20 DNA Artificial Sequence AntisenseOligonucleotide 52 ggcgaagcct gcagcccgga 20 53 15001 DNA M. musculusunsure 11200 unknown 53 tcggtaccta gcacacttgt gggagaaggg aatcataggcaggggcttcc taattgacgt 60 ccacagtcta ttcgtgtata tgtgtggtgc tgggatagaacgctggcaca ctatgcaaga 120 acttaaccct gaaccacgga ctccatccac tcactgggggattctaggta gggactctac 180 ccctgaacca cggaccccag cccctcactg tgcaaagctaggtggaagct cttcctttga 240 gccacaccct agcccctccc tggggcacat ctaggccggactctttcact gagacatagt 300 cctagcattc cacttttttt tttttttttt gaggcagggtggtctgtttt ttgaacttgc 360 taattcaagc tagactctct ggccagtcag ccctagggatcctcctgttt ctgttctccc 420 caattaaagg cacacagttg tagaggaatt ttaatcatggatgttctggc aagggattac 480 attcagagac cttctaggtc catgtgatct ggctggaatgcagacaagtc acaggccatt 540 aatccctctg gctggaatat agacatgcct ttaggacatacctttaatcc caaacgtgta 600 ggtaaagtta gtttgtagaa ggaagaagca tgtttgaattgtgaacctga ttgaggggca 660 agcaaagtga caaatcggga aaatatttga cagaatgactcagaggtagg acatgcccaa 720 ctctcacgtg aaagagaaca gtaggtatgg gtagcagggagacagtttta ttagaacggt 780 tttacagaga gaggttccag gtgaagacag gaaaagctagagaatgagaa ggagccagaa 840 gattagaata gattgccagt gttagtttga agccaagcaaggcaattctg tgagaagcca 900 gtaagcttga agaagagtct gaccagtgaa gcctagctgaaccatccatc caagaactta 960 gaacaagaga gcttattcag tagtaagtct cagaggctgaaatgttctag gcctatatta 1020 gattgtatgg atgctagaag cttccaggcc taggcctggaagtagtttga acagagaaag 1080 aaatgccatg ggctcagccc aaactgtgta ttcgtatatcttgggtacgg ttctcctctc 1140 atccttccat ctgaagaaat aaaagttact tttacacatagtttttttat gtgggtgcta 1200 gaaacttgaa ttcagatcct tgtgtttgga gaacaagtcctttactgact gagccgcttt 1260 gtctgccttc ctggtgacca tttgaatctg tgtccacttcacatgacatc agttgtgcca 1320 tctgtaaaat tgcaccagac tgtttgttgc aagaattgagaggggtttaa tggtagaagc 1380 tgggaagctg ggaggaggga gctggaagga ggaggattgctcaaccttgg tccaggaaac 1440 agtgatcatg tgatcctggt cctgctcctt cccttcctcttatggaggaa gttgtgtgtt 1500 gctccatacg tttgtgaccc tttttttttt tttttttttatcttgttatt cctgccagta 1560 caaatgcaag gaagatagtt ggggagatgg ctctgtggataagagtgagc tcttgatgct 1620 aaagaatgaa gatctgagtg tgaatctcta gcacccatgtgaaaaagctg catggagatg 1680 cgtgctaacc tttagcacca gcagtgagtg agtgggacagagtcagcagg gggcaggggc 1740 ttgttggctc tcaattgagg ttcagcgaga tcgtctggaaagggaatcag gctgcgggta 1800 acaaacccga aacctgatgt tcttctcggg cttctgcatctgtgctgccc cccgcccccc 1860 tggccacgtg tgtcacacac agtcttgcac acacaagataaatttaagga agtgttttgt 1920 aaagttgtgt attttttgga tccttttaac atcagcatactttgttaaat taaggtgaaa 1980 ggaagtggag atgtagcttg gcgaacacaa ggtcagacatagaacagcaa ggagtctaga 2040 gggttaacac agtctattgt tcatggagct ctgtgggaggagcctgtaac cctagcttgt 2100 tagaagtgag agcagcaaca tcaggatgtt ggagctcaagagaatgcttg ctgttggact 2160 gtagagatgg ctcagcagtt aagagcactg actgctcttccagatggtcc tgagttcaac 2220 atccagcaac cagtagtata tgtgtgtgtg tgtttgtgtgtgtgtgtata tatatatatg 2280 tgtgtgtgtg tgtgtatgta tatgtgtgtg tgtatatatatatatatgta tatatatata 2340 tatatatata tatatatata tatatataat cttaaaaaaaatgaatgctt gctgttcttt 2400 tagaagacct atgtttggtt cccagcactc agcatcaagcagccacgcta cctggaactc 2460 cacgtggagg ggatctgatg ctctcttctg gcctctgatggctacagctc ccccccatcc 2520 ccaatacaca cataaataaa atataaatac acataataaaaagaaatcta aacaaattag 2580 attaaggtta ctttgtgctt acccttggct acacggggagttcaaggcca ggtttggata 2640 catgaggcct tgtgaaaaca aacacagcca aacacttcaaaaacaaatgt gggcacaggc 2700 tgctcttgca gaggacctta gttggttctc agcacccacatggtggctca cacacacctg 2760 taactccagt tccaaggaac ctatcactct ctgtggctcccacgggcatg acactcatgt 2820 ggcagacatg tggatagata actagaagta acaagacaaaaaccaaaact agcactgggt 2880 ctctttgtcc catttcccaa tgtgacccca ttgaataagtctttcttttc tgcctttcct 2940 tattaaaaag gaatctttaa acctaacaac agcatgagggctataggtca tactacagaa 3000 aggaggggga tgctagcatt ttgttgcaag tacagtgaaacttgtcaaat gcttaaaaga 3060 ggggaaagaa gataccttcc cgccaggcca gggagaagtgcacagagaag acgagtccat 3120 cctgccattt gatgatcgta gtggggttgt caggctccccctccagagtg atgaacttac 3180 ctgtgagggt cttcataagg atttgcaccc cacctttgagatgaagcagg tatagggaac 3240 atccctccgg gatgttttag tcactctagg catggccatcttctagtttc ttactggcaa 3300 aggtcgatct ctcctggttt gggttggaga tgccttgggtctctgctgtg acatggtggt 3360 ttcgctgggc tcaacttcta gggtgatagg tttgccagttaaggattttt ctttctcact 3420 cgtgtttttt gtatgcatat gtgtgcacat gagtgctggagcccacagag gccagacgag 3480 gacatcacat ctcctggacc tggagtttat aggtgtctgtcagccccgtg atgtgggtgc 3540 tggagatgaa ttctgccaga gcagtaagca gtcttaacttctgagccatc attgtcagtg 3600 atggtggtag cagtcctcac agaaacacag ctctgatctcaatggatata actccaatat 3660 cagacttctg aggcagggtt tcatgtagct caggctgaccgagaactccc tatcgtagct 3720 aaggatgacc ttagactcct gagcctcagg cttctacctcacaaatgatt atgattacag 3780 ctgtgtgctt tgccctgctt aatgatactg tattgtgggctgaccagatg gctcactggg 3840 taaaggaact tgaagccaag catgaggagc tcaggtaaatctcttgggac ccacatggta 3900 gaagaaacct gtggaaacct gataagcttt ctgctatatttactgaggta ggctctgtta 3960 ctgaacccga aacaagattg ggggagggct agatcctctgtctctgtctc atgtttttgt 4020 ttgtttgttt tttgtttttt ggtgtttgga gacagggtttctctgtatag tcctggctgt 4080 cctggaactc actttgtaga ccaggctgtc ctcaaactcagaaattcacc tgcctctgcc 4140 tcccgagtgc tgggattaaa ggcatgcgcc accacacccgactctctgtc tcatgttttg 4200 atcacaggag gccaccatac ctgcccatct tttatgtggtccccttgcct gtagagcagg 4260 taccacatcc attgagtcat gtctcagccc ctgaacccattttcatccag tcatctggtg 4320 acaagcatct aggctgctcc tatttcttgc ctgctggtttggcacagggt cttgtgcagc 4380 ctatgtgcct gggttcctga cagtcaggtc caaaagaaaccgaggacaaa tagctgactc 4440 tgaagtctat tatcatacca aagtgcacgc tggcctgaatactctttcag taccaccagc 4500 acctgttaca tcttctctgc tcttctcacc tcacccttaccctaaacttc ttcagctaat 4560 agtcttctcc tagcttttta tccctatagt actctgccatttcagccatg catcctttgg 4620 atttggttct ctctctctct ctctctctct ctctctctctctctctctct ctctctctct 4680 ctctctctct ctctctctct ctctcggatc tctctcctctttctctctca ttctcccctg 4740 ctgtctttcc tctaatggcc cagctcggtt tactgaacatgtttagtctc tctctccctc 4800 tgctctgctc tgtctgtctg tctgtctgtt tctccctgctctggaccctt ctagatgtct 4860 ctggctgtac tctcccttgt atctacaata acacccccccccaatgccag tgtactggtc 4920 atgtcctcag tgtatacatt ggccatccta cctccaccatccatacctct caagtgacag 4980 gcatgtcaca ctaggcaagc actctaccaa ctgagccgcattcccaacct cagattttct 5040 tagtcagtat ttttttttta atattagcaa ataaattagcagttttcagg gtgggagaaa 5100 tatcacagtg gtgaagaaca cttgctgctc ttggagctgatggaagttga gttcccagca 5160 cctacacaag acagctcatc cacctctaac tcagtactagagaatcttat tccctcctct 5220 gacctcctag ggcacagaca caagcacgga acacatacagagggatatat gtaaacaata 5280 aaaataattt taccaaatgc tgggtttcat tatgacattatcacacacat atatcagtat 5340 ccgtagctca tatttacctt ccattaacct ccttgtccctcctccctctc ctactggtcc 5400 catgcagacg tgtgtgtgtt atttatgtat ccgtcctcctactgtgagag aaaacatgtg 5460 atgttgtctt tttttcatta cctctcttgt tgtcccctcccctcagtcca ttccacatgt 5520 aattctactt tcaagactta tagatatttt acatctgaacttcacgtgag agaggacacg 5580 tgacacttgc tttctgagtc ttgcttgctt tgcctaacatggtgctgtct agttccattc 5640 atctttctgc aggtgaaatc tcttcattct tctccacagcttcattcacc attccgtagt 5700 gcatagatac catgctttct ttatctgttt atctgctggttccattcctc agccactgtg 5760 aacaatgcag cagtaaacat ggatgtgcat gtatgcacatggctggctga ctgactcctc 5820 taggtatata tagagtggta tagctggatc atatgctagttctaatttta attgctttaa 5880 ttcctttgct gtgtatgcat acacacgtgc acatgtgcatgtatgcactc agataaggtg 5940 tgagagcggg cacgtgtgac ctatagatgt cagagaacagctttgtgaag ttggttccct 6000 ccttccactt cctgtgggtt ctagagattg gcatgagatcaggcttgtgc agcaaaccat 6060 cttgctagct ctgattataa ttaaaaaaaa aaaacttttatagtggactg gagaaatgac 6120 tcagttgtta agagcattca ctgttcttag agaggaccagggttcaattc ccggcacaca 6180 tgtggtggat cacaactatc agtagttacg catgagttgtacacacatac acgagggcaa 6240 cgcactcatg tacataaaat aaaaataagt aaatctttaaaaaaaaagct tacatataca 6300 ctggtggaag tgtatctggg ggaaaagaga ccgagaagatagcccacaaa acaagcatga 6360 gggcctgaat tcagatcccc agccacctca taaaaacccatgggcatgtg aggcaaaggc 6420 agacggatca ctccaagttc aaggtcatcc tggtgtacatagcaagtttc tagccagcca 6480 gggctaccta gagaatcctt ttctcaaaaa gaaaaaagaaagtgaaaagc gattaaggaa 6540 gatgtacaat gttaatctct agattccaca tgcgccacacacaggagccg ggtgagacta 6600 ggggtatatc tcagagatag aacactccct agcctataaggaccctaagt tccattccta 6660 gcacaaaggt gggggatggt aatgatttta tgataatatttccataactt tcctttgtct 6720 tttgtatgta ttactgtctg tgtgcattca gccagttcaaaacgcagaca gttgacatgt 6780 ttccagccag ttaagtaagt taacatgggc ctcctgttctgtggttaccc actctctaca 6840 tgtgcataca gcaggagaat ccaacacttt tcaacagaaactccaattac cagacagcat 6900 caagaattct cctttgattg acagaaatat ctgtcatagcaaccttgtca ccagggcaac 6960 aagttatcat tgaacacaca gccagtcaga agcttcatttctggggaaga tagcaacagc 7020 ttccaagtgg tcctagccac ttttgtccaa atgaggcatcatggacagag gggtgtcagg 7080 ggggagagta agccagcaag agttctactg ttgaaccacattgtacatct cacccctggt 7140 tcctctcatt ctgatgacag aactgagagg ctatgacagaccccatggac acaaaaccag 7200 gtgcttttac atttgacctt tgaaaggaag tttctcacctcaggtctcca gcaatgcctg 7260 ggaatggaga cccccaaagc agctgtgtac tatgttggcgatggccacta aagtcgcatg 7320 aacacagaag ccaggtggga ggaagagcac atcacctgaatccacttatg ctgaagagga 7380 aagagacaaa tcttgcacgg ggtgggggtg gggaggcttagttcagttag agtgcctggc 7440 aggcacaaag tcctgtgctc aagtcctcga agtacagaaaccatgcacag tggtgcgtcc 7500 ctgccacccc agcatcgaga atgcaggatg gtcaggagttcaaggtcgtt cttggctaca 7560 cagttcaaac ttgcatagct tacaaaagag cttggattcaatgaataaat agatagataa 7620 ctaaataaag attaaagtct tttcatttgt ccaatttataaacttcctct gctaatctca 7680 attttgtcct atctagttaa aagacatcga ggccagtgtaacagcttcag agccggacag 7740 cctgacgtta acacccagga tttgcatagc cacctccagttgtcctcacc tccgtatgca 7800 catcacactg tggcgtgcac gcacttacac ccacacgtgtcacgcacatg caataatgac 7860 taaataatgt ttttaaagac agagaagata aggcagattctattgagtca ttcacacaca 7920 taaatattct gtatgtgggc cggagctatg actacccaagatttattatt ttaattactt 7980 gttgagaaag agggatttcc aaagggacag gccccttgctccttgcacca gaaagcagaa 8040 agcagaaagc agaaagcaga aagcagaaag cagagctcttgcctagactc ctccagtgag 8100 atgttttggg taatggctgg gaacctcctt tctcagtgggtcctttctca gtggtagagc 8160 tcttgcttag ccccaccccc accccgcccc cagagaggggctgggtctct agctctgtgg 8220 tagaccatct gtatagagca taccactgag aggctggagacatggttcag aggtagggta 8280 cctggctgga attccatagt gagggtctgg tggtgtggttcagcaaggga gcagttgact 8340 agattttagc tggtagagct tgcctacact gtatatccccgccaaacata gaaacataca 8400 tgacagctgt ggtcacacag ggaccgtgtc tcagtttgcatctccagccc tacaggtgaa 8460 gtcttggctc ctagccttgc ccctcccacc atggtccctggtgttatctc cagacaacgt 8520 cctgagaaga caatccttct aatgtaggaa gggctgagcaggagggaaat ccctcctctg 8580 ggacctctag agttcacctc tcttggggtc agatgtggcaatggaaggcc aggggccagg 8640 gtacttatta tccaaggcag gatgggccag aagtcaacaagaaggagctg tgggacagga 8700 gctaataccc agaactgagt tgtgtcctgc taagtcctctgccacggtaa gtgatagcca 8760 tcccccagtc agagtgctga tgtggcggcc ccagagctgctgaatgtcca tccatgtgtg 8820 ccctcaggaa ctaaaggaca ttgcagtctg actcccccagcatgggaagg gtctggtcta 8880 aggcggggct cttgcttgtc tcttgcagta cccacgggatgaagaacctt tcatttcccc 8940 tccttttcct tttcttcctt gtccctgaac tgctgggctccagcatgcca ctgtgtccca 9000 tcgatgaagc catcgacaag aagatcaaac aagacttcaactccctgtgt gaggatcccc 9060 ccttcgcccc catccaacct tgccgagggc cttacagacttatccctgat ggggaacaga 9120 cccgcccagc tacaaggcta ccctcagcct cagcctcaccctgttccaac ccaaattcat 9180 cctgacctac cttgatcatg gttcacccca caacttagcttcttccaacc atcacctcca 9240 aacctcccct cactccaaaa ggccccacac tgtggtccatggagctgagc ccccagtgcc 9300 tgaccctctt gcctcagcct cctgagtgtt tggagtacaggtgtgagcaa aatattttac 9360 cgtgaacgtg tgctgtccat gtctgtgtat gtgtgtttatatatgtggtg cacatatgtg 9420 aaggagcttg tgtgtgcata ggcatatgga gaattgaagttggtgtagtg ttatcattcc 9480 acctctgctt tgttgagaca gaatctctca gctgaacccagggcttgtgg gtttgtggta 9540 gtctagttag ccatcttgtt ctggggactc catgtctgtctgcctcctgg aggctgggat 9600 tatgggtgcc cctacaccat gcatgcctag cttctaaatgggttctgggg atcttggctc 9660 tgatcctccc acttgtgagt caaactcttt agctataagtcaccttctca ctctgctgag 9720 aagaatttga cctcactgtc agtgtcaaac ccaaagatcgccccctgcaa agatgcagct 9780 ggattacccc ttcaatcttc ccttctgcag ttccaaatgcaataaagaac attggcttaa 9840 attgctggac agtctcctcc agagggaagt tggcctcctgcccagaaggt gagtgcaaac 9900 tctgttgttc agactttctg gaagttctga aactcctctgtctgccttgt ctgggtcctc 9960 aacatgtctg tccttaggac tccctcccag acttccagtcttctcatctc ccagcccagc 10020 tcaaggtcct ttagactcct gctgtatcat cttgcactctgtttccgtgg caacagcctc 10080 cagccccctc ccccatctct gcctcccacc tgccacagtcttccggtttt tcagggactt 10140 ctttccttgc aggcacagca gtcttgagct gctcctgtggctctgcctgt ggctcgtggg 10200 acattcgtga agaaaaagtg tgtcactgcc agtgtgcaaggatagactgg acagcagccc 10260 gctgctgtaa gctgcaggtc gcttcctgat gtcggtcagttgagaactga gcttgtcctt 10320 cttatgttga cagggacggg gaggaggcag gggcaggtctaagctgaggg tctggaaatg 10380 aagaatgaga agatggtgat gatgaagatg gatggcgaagtgggcatggg ccttgtgtga 10440 cttctttagt ggagaagggt gtgtgtggga attgtgtgggaaatgttgca cagtgttgtg 10500 aattttacta cttgtccatc actccttttt tcttctcttccccttcccct tcctctttga 10560 gactctcata tagtccaggc tggcctcaaa tgatagtcaccatcacagag gagactggac 10620 cttggcagga ctgaggttcc atagctctca agttcacttttcacctctgt ggatatgatg 10680 tcgaatacct attttcaacc agaggcaccc agggcatttacaccacctgg ggcagaggat 10740 atcgttttcc ttgtatcttg agcttagaat aaggtatagcctgggtgcta gtaactgtct 10800 atagaatgag ggctggtgag atgattcagt gggtaaagatgctgctgcca aggctgatga 10860 tcagggcttg attcctggaa actacatggt ggaaggagagagtccaggtt gtcttcccac 10920 ttccacatgg gcactgaggc acttgagtac cacgtgtgcacacacagtga atgaaagagt 10980 gaatgaatga agcaataaaa gtatctgatg agcctggcatggtgacacat gcctttaatc 11040 aaagcacttg caaggcagag acaggtggat ctctgtgttcaaggccagct ggtctacaga 11100 gaaaaaaaat ccaaaagaaa gagaacgaaa gaaagagagagggagggagg gagggaggag 11160 ggagggaggg agggaggagg aaggaaggag gaattcaaannnnnnnnnnn nnnnnnnnnn 11220 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn 11280 nnnnnnnnnn nnnnnnnnct ttctcttccc ttcctgtccctcctgatcag caccaggagg 11340 gaggaaggaa ggaaggaagg aatcaaatta gtaactgcaatggttaacag atctagaatt 11400 acctagaaga cacgtatctg ggcatgcctg gaagggagttggttaattgg tttaattcaa 11460 gcgggaggac cctgattgaa taaaaaggag aaagtgggagttggagagaa ttcagagaat 11520 taagaacact tgctgctcct gcaagggacc caacccagttttccagtacc tagtttggac 11580 agctgacagt catctttaac tcatatgccc atacacacttatacaccccc cccacacaca 11640 cacacacact cacacactca cacatactca cacacacacacatactcaca cacacattca 11700 cacacacatg cacacgtatg caaaccagca atggcacctgcagagatggc tcagaagtta 11760 agagcacttg ctgttcttcc agaggactga gttcatttcccagcactcaa atgaagtgtg 11820 tcttagttag ggttaccatg accaagaaac aagttggagaggaaagggtt tatttagctt 11880 acacttccat actgctgttc atcattgtag gaagtcaagacacaaactca cacagggcaa 11940 gaacctgtag gcagagctga tgcagaagcc atggagggatgctgctcact gacttgtggc 12000 ccctgctttg ctcagcctgc cttcctatag aacccaggaccaccagccca gagattgcac 12060 tagacacaat gagctgggtt ctctgccact gatcagtaactgagaaaatg ccctgtagtg 12120 gatcttatga aggtatttcc tcaattgagg cacttcctctgatgactcta gcttgtgtca 12180 atgagacgca aaaccagcca gtacagagtg actcaagaccatctgtatct ttggctacag 12240 gagatttgac acctctgacc agtgcatgca cctgtacttatgcacaaata cccacattta 12300 gatacaaaca ttccttcaca taatcaaaaa gaatacaatataagccgggt gtagaggcac 12360 acatcttaaa tcctagcact cgggaggcag aggcaggtggatttctgagt tcaaggccag 12420 cctgattctc agagtgagtt ccagaacagt gaaaactacacagagaaacc ctgtcttgaa 12480 aaaaaaaaaa aaacaaacaa ccagaaaaca aagaaacaaagaaacaaaaa ccaaccaaaa 12540 aagaaaaaaa gaaaaaaaaa aaaggaagaa aaatttaggggccagcaaga ggctcagtgg 12600 gtagaggctc ctgctaccaa gctgaccatt tacacatatgtacacaaaat aaaataaatt 12660 taaatgtaaa aaataaaaat tggggcctaa ggaaagatgggtcaatggtt aagagcatgt 12720 attgcttttt caggggaagt gagcgtggtt tccagcacagccacccgttc ctgtagctcc 12780 agagatgtct gatgtcctcc ggtctctaca ggcacctgcactcacgtgcg cgaatccaca 12840 cacaagcaca catacttaaa aataaaacaa aacaggctggatgtggtggt ctcatacttt 12900 taattccagt acttgggaac tagagacaga ctgttagtttatggccatcc tggcttgggg 12960 gatggctcag tggttagcag cactgactgc ccttctaaaggttcaatccc aagccatcag 13020 taatctatgt cgggctggag aggtggctca gcggttaagagtactgactg ctcttcctga 13080 ggtcctgagt tcaattccca gcaaccacgt ggtggctcacaaccatccta tgcccccttc 13140 tggtgtgtct gaagatagct acagtgtact catatacaaaaaataaataa taataataat 13200 ttttttaaaa aggtcatcct ggtctaccta gccagtgctacatagttcca atgaattaag 13260 aacaaatgga atattttaaa ttgtctttga tattgaaagttgttctcttt aattttttat 13320 ttattcactt tacatcctgc tccactgcca cccttctggttgccccgttc cacaatcatt 13380 ctcccatctc ctcctcctct aagtgtgtgg ggccccctaggtatacccct accctgttac 13440 ttcaagtctc tgtggggcta ggcacttcct ctcccactgaggccagcaga ccagacagct 13500 cagctagaag actacagccc acgtacaggc aacagcttttggaatagctc ctgctccagc 13560 tgtttgggac ccacatgaag acccaagctg cccatctgatatatatgtgg ggggaggcct 13620 aggtccagtc tattatattt tttggttttg tttgtctctgggtatcttca aatgtctgtc 13680 cagcccgaag atttcaaagg aaataaatct ctggccagagtttaacacac cacaataaac 13740 ctaaatccac ctagagtcct ccaccagttt catggttgaaccttcaccac agccaattag 13800 agagagtagc agataagcca acgatgagaa gtgaagacagcgggaatgtc cagtcagttg 13860 tgggtctcat cctgctcttc ttcctgggtg agagtgcagctctgacagac actgagactc 13920 acacagtgga tggttttctc agaggaagaa gacatatgcccactggcact tgagtctgtt 13980 actggctgtc gtgatgggca atgcaattca ccatttcccagggagcaaga acggcaagca 14040 cctttgtgta tccagttctg cctctctcca ttgcgcataaaccgctttcc ttcatctttc 14100 atttcctcac tgcacacttg ttagcgtcta ccacctacttactcacttcc tggtgcaact 14160 gtgcctggcc agggctgggc tcagggttgt ggtattgtcctggtacttcc tgacgcacca 14220 gggtggaaag ttgttttcat acagtatatt ctgatcaggctttcccttcc tatctcctcc 14280 catcttccca cccatgaacc cgcccttctc tctcaaacaggcagacaaga aacaaacaaa 14340 caaacaaaca aacaaaaaac tgaaaacaaa aaaaacccaccaacctcccc ccccaaaccg 14400 gcataataat ttaaaaagaa aaagtacaac aacacgcacgcacacacaca cacacacaca 14460 tacactcaaa caaccaaacc aactcaaaac cccaaaacttaaaaaaaaac ccaaacccat 14520 aaaaccccac aaaattggga accataatgt ataagcaaaaggcccagtaa acataacaca 14580 ttacaaaaaa ttatgaaaca aaaactttac taagtgttgttcataaagcc tataagattc 14640 ctttgaagta aactaatttt tcctttgcaa gccattgttgtcaagtgatt aggggtggct 14700 gcccatgtcc acttccccct ctctgtgctg ggactccggctggctcgaac ctgcacaggt 14760 cctgtgctgc tgctacagtc tctgcgagtt catgtgtgtgtgagtcttgt atagcttgga 14820 agacactgtc tccttggtgc catccctagt tcttatgatctcttctcttc tgtgtagctt 14880 cctggatcct gatggggagg gtctgatgaa cacattccatataggactaa gtgtactaaa 14940 atctatcact ccacgcactg tccagtaaag ggtactaagtaagctcccat ctgctgcaag 15000 a 15001 54 20 DNA Artificial SequenceAntisense Oligonucleotide 54 attagctcct gtcccacagc 20 55 20 DNAArtificial Sequence Antisense Oligonucleotide 55 gggtattagc tcctgtccca20 56 20 DNA Artificial Sequence Antisense Oligonucleotide 56 actcagttctgggtattagc 20 57 20 DNA Artificial Sequence Antisense Oligonucleotide 57ttagcaggac acaactcagt 20 58 20 DNA Artificial Sequence AntisenseOligonucleotide 58 gaggacttag caggacacaa 20 59 20 DNA ArtificialSequence Antisense Oligonucleotide 59 ggcagaggac ttagcaggac 20 60 20 DNAArtificial Sequence Antisense Oligonucleotide 60 tgggtacgtg gcagaggact20 61 20 DNA Artificial Sequence Antisense Oligonucleotide 61 tcatcccgtgggtacgtggc 20 62 20 DNA Artificial Sequence Antisense Oligonucleotide 62aaggttcttc atcccgtggg 20 63 20 DNA Artificial Sequence AntisenseOligonucleotide 63 ggaaatgaaa ggttcttcat 20 64 20 DNA ArtificialSequence Antisense Oligonucleotide 64 gttcagggac aaggaagaaa 20 65 20 DNAArtificial Sequence Antisense Oligonucleotide 65 agcccagcag ttcagggaca20 66 20 DNA Artificial Sequence Antisense Oligonucleotide 66 ctggagcccagcagttcagg 20 67 20 DNA Artificial Sequence Antisense Oligonucleotide 67gcatgctgga gcccagcagt 20 68 20 DNA Artificial Sequence AntisenseOligonucleotide 68 cagtggcatg ctggagccca 20 69 20 DNA ArtificialSequence Antisense Oligonucleotide 69 atcgatggga cacagtggca 20 70 20 DNAArtificial Sequence Antisense Oligonucleotide 70 cttcatcgat gggacacagt20 71 20 DNA Artificial Sequence Antisense Oligonucleotide 71 tcgatggcttcatcgatggg 20 72 20 DNA Artificial Sequence Antisense Oligonucleotide 72gatcttcttg tcgatggctt 20 73 20 DNA Artificial Sequence AntisenseOligonucleotide 73 gggagttgaa gtcttgtttg 20 74 20 DNA ArtificialSequence Antisense Oligonucleotide 74 acagggagtt gaagtcttgt 20 75 20 DNAArtificial Sequence Antisense Oligonucleotide 75 gaaacaggga gttgaagtct20 76 20 DNA Artificial Sequence Antisense Oligonucleotide 76 gcatttggaaacagggagtt 20 77 20 DNA Artificial Sequence Antisense Oligonucleotide 77ttattgcatt tggaaacagg 20 78 20 DNA Artificial Sequence AntisenseOligonucleotide 78 ctttattgca tttggaaaca 20 79 20 DNA ArtificialSequence Antisense Oligonucleotide 79 gccaatgttc tttattgcat 20 80 20 DNAArtificial Sequence Antisense Oligonucleotide 80 caatttaagc caatgttctt20 81 20 DNA Artificial Sequence Antisense Oligonucleotide 81 gcaatttaagccaatgttct 20 82 20 DNA Artificial Sequence Antisense Oligonucleotide 82tgtccagcaa tttaagccaa 20 83 20 DNA Artificial Sequence AntisenseOligonucleotide 83 gagactgtcc agcaatttaa 20 84 20 DNA ArtificialSequence Antisense Oligonucleotide 84 ttccctctgg aggagactgt 20 85 20 DNAArtificial Sequence Antisense Oligonucleotide 85 gccaacttcc ctctggagga20 86 20 DNA Artificial Sequence Antisense Oligonucleotide 86 ggccaacttccctctggagg 20 87 20 DNA Artificial Sequence Antisense Oligonucleotide 87caggaggcca acttccctct 20 88 20 DNA Artificial Sequence AntisenseOligonucleotide 88 gggcaggagg ccaacttccc 20 89 20 DNA ArtificialSequence Antisense Oligonucleotide 89 actgctgtgc cttctgggca 20 90 20 DNAArtificial Sequence Antisense Oligonucleotide 90 cacaggagca gctcaagact20 91 20 DNA Artificial Sequence Antisense Oligonucleotide 91 gtcccacgagccacaggcag 20 92 20 DNA Artificial Sequence Antisense Oligonucleotide 92atgtcccacg agccacaggc 20 93 20 DNA Artificial Sequence AntisenseOligonucleotide 93 cttcacgaat gtcccacgag 20 94 20 DNA ArtificialSequence Antisense Oligonucleotide 94 ctttttcttc acgaatgtcc 20 95 20 DNAArtificial Sequence Antisense Oligonucleotide 95 cactggcagt gacacacttt20 96 20 DNA Artificial Sequence Antisense Oligonucleotide 96 tgcacactggcagtgacaca 20 97 20 DNA Artificial Sequence Antisense Oligonucleotide 97atccttgcac actggcagtg 20 98 20 DNA Artificial Sequence AntisenseOligonucleotide 98 gtctatcctt gcacactggc 20 99 20 DNA ArtificialSequence Antisense Oligonucleotide 99 tgtccagtct atccttgcac 20 100 20DNA Artificial Sequence Antisense Oligonucleotide 100 ctgctgtccagtctatcctt 20 101 20 DNA Artificial Sequence Antisense Oligonucleotide101 cagcgggctg ctgtccagtc 20 102 20 DNA Artificial Sequence AntisenseOligonucleotide 102 acagcagcgg gctgctgtcc 20 103 20 DNA ArtificialSequence Antisense Oligonucleotide 103 cctgcagctt acagcagcgg 20 104 20DNA Artificial Sequence Antisense Oligonucleotide 104 cgacctgcagcttacagcag 20 105 20 DNA Artificial Sequence Antisense Oligonucleotide105 ggaagcgacc tgcagcttac 20 106 20 DNA Artificial Sequence AntisenseOligonucleotide 106 tcaggaagcg acctgcagct 20 107 20 DNA ArtificialSequence Antisense Oligonucleotide 107 tccccgacat caggaagcga 20 108 20DNA Artificial Sequence Antisense Oligonucleotide 108 ctcacttccccgacatcagg 20 109 20 DNA Artificial Sequence Antisense Oligonucleotide109 gctggaaacc acgctcactt 20 110 20 DNA Artificial Sequence AntisenseOligonucleotide 110 ctgtgctgga aaccacgctc 20 111 20 DNA ArtificialSequence Antisense Oligonucleotide 111 tacaggaacg ggtggctgtg 20 112 20DNA Artificial Sequence Antisense Oligonucleotide 112 catctctggagctacaggaa 20 113 20 DNA Artificial Sequence Antisense Oligonucleotide113 gacatctctg gagctacagg 20 114 20 DNA Artificial Sequence AntisenseOligonucleotide 114 atcagacatc tctggagcta 20 115 20 DNA ArtificialSequence Antisense Oligonucleotide 115 aggacatcag acatctctgg 20 116 20DNA Artificial Sequence Antisense Oligonucleotide 116 agaccggaggacatcagaca 20 117 20 DNA Artificial Sequence Antisense Oligonucleotide117 tgcttgtgtg tggattcgcg 20 118 20 DNA Artificial Sequence AntisenseOligonucleotide 118 tgtgcttgtg tgtggattcg 20 119 20 DNA ArtificialSequence Antisense Oligonucleotide 119 tatcacttac cgtggcagag 20 120 20DNA Artificial Sequence Antisense Oligonucleotide 120 atggatggacattcagcagc 20 121 20 DNA Artificial Sequence Antisense Oligonucleotide121 cccgtgggta ctgcaagaga 20 122 20 DNA Artificial Sequence AntisenseOligonucleotide 122 acgttcacgg taaaatattt 20 123 20 DNA ArtificialSequence Antisense Oligonucleotide 123 gattctgtct caacaaagca 20 124 20DNA Artificial Sequence Antisense Oligonucleotide 124 cccagaacccatttagaagc 20 125 20 DNA Artificial Sequence Antisense Oligonucleotide125 actgctgtgc ctgcaaggaa 20 126 20 DNA Artificial Sequence AntisenseOligonucleotide 126 ctcaactgac cgacatcagg 20 127 20 DNA ArtificialSequence Antisense Oligonucleotide 127 cataagaagg acaagctcag 20 128 20DNA Artificial Sequence Antisense Oligonucleotide 128 ctccatggcttctgcatcag 20 129 20 DNA Artificial Sequence Antisense Oligonucleotide129 cataagatcc actacagggc 20 130 20 DNA Artificial Sequence AntisenseOligonucleotide 130 taaccattga cccatctttc 20 131 20 DNA ArtificialSequence Antisense Oligonucleotide 131 ctcacttccc ctgaaaaagc 20 132 20DNA H. sapiens 132 cctgcaggat gaaagctctc 20 133 20 DNA H. sapiens 133gaaagctctc tgtctcctcc 20 134 20 DNA H. sapiens 134 gaagaagcca tcaatgagag20 135 20 DNA H. sapiens 135 caatgagagg atccaggagg 20 136 20 DNA H.sapiens 136 ggatccagga ggtcgccggc 20 137 20 DNA H. sapiens 137aggaggtcgc cggctcccta 20 138 20 DNA H. sapiens 138 aggtcgccgg ctccctaata20 139 20 DNA H. sapiens 139 gcaataagca gcattggcct 20 140 20 DNA H.sapiens 140 ataagcagca ttggcctgga 20 141 20 DNA H. sapiens 141cagcattggc ctggagtgcc 20 142 20 DNA H. sapiens 142 ctgtggctcg tgggatgtgc20 143 20 DNA H. sapiens 143 tcactgccag tgcgcgggca 20 144 20 DNA H.sapiens 144 gtgcagccct gaggtcgcgc 20 145 20 DNA H. sapiens 145gctccaggtc cggaggggtt 20 146 20 DNA H. sapiens 146 ggggagctgg aaataaacct20 147 20 DNA H. sapiens 147 ctggaaataa acctggagat 20 148 20 DNA H.sapiens 148 aataaacctg gagatgatga 20 149 20 DNA H. sapiens 149acctggagat gatgatgatg 20 150 20 DNA M. musculus 150 agtcctctgccacgtaccca 20 151 20 DNA M. musculus 151 tgccactgtg tcccatcgat 20 152 20DNA M. musculus 152 ttggcttaaa ttgctggaca 20 153 20 DNA M. musculus 153ttaaattgct ggacagtctc 20 154 20 DNA M. musculus 154 tcctccagagggaagttggc 20 155 20 DNA M. musculus 155 aaagtgtgtc actgccagtg 20 156 20DNA M. musculus 156 tgtgtcactg ccagtgtgca 20 157 20 DNA M. musculus 157gccagtgtgc aaggatagac 20 158 20 DNA M. musculus 158 gtgcaaggatagactggaca 20 159 20 DNA M. musculus 159 aaggatagac tggacagcag 20 160 20DNA M. musculus 160 gactggacag cagcccgctg 20 161 20 DNA M. musculus 161ggacagcagc ccgctgctgt 20 162 20 DNA M. musculus 162 aagtgagcgtggtttccagc 20 163 20 DNA M. musculus 163 cgcgaatcca cacacaagca 20 164 20DNA M. musculus 164 cgaatccaca cacaagcaca 20 165 20 DNA M. musculus 165tgctttgttg agacagaatc 20

What is claimed is:
 1. A compound 8 to 80 nucleobases in length targetedto a nucleic acid molecule encoding resistin, wherein said compoundspecifically hybridizes with said nucleic acid molecule encodingresistin and inhibits the expression of resistin.
 2. The compound ofclaim 1 which is an antisense oligonucleotide.
 3. The compound of claim2 wherein the antisense oligonucleotide comprises at least one modifiedinternucleoside linkage.
 4. The compound of claim 3 wherein the modifiedinternucleoside linkage is a phosphorothioate linkage.
 5. The compoundof claim 2 wherein the antisense oligonucleotide comprises at least onemodified sugar moiety.
 6. The compound of claim 5 wherein the modifiedsugar moiety is a 2′-O-methoxyethyl sugar moiety.
 7. The compound ofclaim 2 wherein the antisense oligonucleotide comprises at least onemodified nucleobase.
 8. The compound of claim 7 wherein the modifiednucleobase is a 5-methylcytosine.
 9. The compound of claim 2 wherein theantisense oligonucleotide is a chimeric oligonucleotide.
 10. A compound8 to 80 nucleobases in length which specifically hybridizes with atleast an 8-nucleobase portion of a preferred target region on a nucleicacid molecule encoding resistin.
 11. A composition comprising thecompound of claim 1 and a pharmaceutically acceptable carrier ordiluent.
 12. The composition of claim 11 further comprising a colloidaldispersion system.
 13. The composition of claim 11 wherein the compoundis an antisense oligonucleotide.
 14. A method of inhibiting theexpression of resistin in cells or tissues comprising contacting saidcells or tissues with the compound of claim 1 so that expression ofresistin is inhibited.
 15. A method of treating an animal having adisease or condition associated with resistin comprising administeringto said animal a therapeutically or prophylactically effective amount ofthe compound of claim 1 so that expression of resistin is inhibited. 16.A method of screening for an antisense compound, the method comprisingthe steps of: a. contacting a preferred target region of a nucleic acidmolecule encoding resistin with one or more candidate antisensecompounds, said candidate antisense compounds comprising at least an8-nucleobase portion which is complementary to said preferred targetregion, and b. selecting for one or more candidate antisense compoundswhich inhibit the expression of a nucleic acid molecule encodingresistin.
 17. The method of claim 15 wherein the disease or condition isa metabolic disorder.
 18. The method of claim 17 wherein the metabolicdisorder is diabetes.
 19. The method of claim 17 wherein the metabolicdisorder is obesity.
 20. The method of claim 15 wherein the disease orcondition is atherosclerosis.